KR20200103703A - Multispecific chimeric receptor comprising NKG2D domain and method of use thereof - Google Patents

Abstract

The present application provides a chimeric receptor targeting NKG2D, and a multispecific chimeric receptor comprising an NKG2D domain and a second antigen binding domain such as an IL-3 domain. A dual chimeric receptor system is also provided comprising a chimeric receptor comprising a first NKG2D domain, and a second antigen binding domain such as a second chimeric receptor comprising an IL-3 domain. Additionally, engineered immune effector cells for cancer treatment (such as T cells), pharmaceutical compositions, kits, and methods of treating cancer are further provided.

Description

Multispecific chimeric receptor comprising NKG2D domain and method of use thereof

Cross-reference to related application

This application is an international patent application number filed on December 28, 2017. Claims the benefit of the priority of PCT/CN2017/119397, the contents of which are incorporated herein by reference in their entirety.

Submitting a sequence listing as an ASCII text file

The contents submitted as follows as ASCII text files are incorporated herein by reference in their entirety: Computer-readable form of sequence listing (CRF) (file name: 761422000941SEQLISTING.txt, date of record: December 28, 2018, size) : 85 KB).

Field of this application

The present invention relates to chimeric receptors, multispecific chimeric receptors, dual chimeric receptor systems, engineered immune effector cells, and methods of use thereof.

Background of the invention

Acute myelogenous leukemia (AML) is characterized by abnormal clonal proliferation of myeloid precursors that dominate the bone marrow and blood, which severely impairs normal hematopoiesis. AML is the most common type of acute leukemia, accounting for about 25% of all adult onset leukemias in the Western world. AML occurs in 3-5 per 100,000 adults annually, with the highest mortality rate among all leukemias (DiNardo and Cortes 2016). Over the past 40 years, the 5-year relative overall survival rate of AML patients has increased slightly from 6.3% between 1975 and 1980 to 23.9% between 2007 and 2012 (Mardiros et al. 2015).

The standard first line therapy for AML is chemotherapy using cytarabine with anthracycline as an induction therapy, followed by high-dose cytarabine and/or allogeneic stem for patients who have achieved complete remission (CR) after this induction therapy. It uses repeated cycles of cell transplantation (alloSCT). Almost 70% of young patients can achieve initial CR with current induction chemotherapy, but 43% of patients eventually relapse, and 18% never get CR even with first-line induction therapy (Forman and Rowe 2013). ). AlloSCT is the preferred method of treatment after the second remission. The 5-year disease-free survival rate among patients receiving alloSCT is 40-50%, showing the sensitivity of AML to immune-based therapy. However, patients with primary refractory disease or with an initial CR less than 6 months have little benefit from alloSCT treatment (Mardiros et al. 2015). In addition, cytotoxic damage to the patient's organs by conventional salvage chemotherapy further impairs the likelihood of success of alloSCT. Therefore, after relapse or induction failure, more effective and less toxic therapeutic agents are needed for AML patients.

T cells have the potential to attack and eradicate tumors, particularly those with a high mutagenic load that results in neo-antigens. However, the antitumor capacity of T cells is often actively inhibited by the immune-suppressive tumor microenvironment (TME) (McGranahan et al. 2016). Chimeric antigen receptor T cells (CAR-T) are an extracellular antigen-binding domain directed to an antigen on a tumor cell, a hinge region, and an intracellular T cell receptor (TCR) to induce T cell activation upon antigen binding. It is constructed by transducing (signaling) a gene encoding a fusion protein, including a signaling domain. Unlike conventional T cells that rely on native TCRs for tumor antigen recognition, CAR-T cells are redirected to unprocessed antigens, whereby tumors are independent of the expression of major histocompatibility complex (MHC) antigens. Kills cells Thus, CAR-T cells have the ability to overcome many inherent limitations of immunotherapy. After 20 years of preclinical studies and clinical trials, the safety and feasibility of CAR-T-based therapy was confirmed, and unprecedented clinical results were obtained in hematologic malignancies (K℃henderfer et al. 2015; Louis et al. 2011).

The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are incorporated herein by reference in their entirety.

Brief summary of this application

The present application provides a multispecific chimeric receptor and dual chimeric receptor system targeting an NKG2D ligand and a second antigen, such as a tumor antigen such as CD123. Chimeric receptors targeting the NKG2D ligand are also presented.

One aspect of the present application provides a chimeric receptor comprising: (a) an extracellular domain comprising an NKG2D domain; (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the extracellular domain comprises a first NKG2D domain and a second NKG2D domain.

One aspect of the present application provides a multispecific chimeric receptor comprising: (a) an extracellular domain comprising an NKG2D domain and a second antigen binding domain; (b) transmembrane domain; And (c) an intracellular signaling domain.

One aspect of the present application provides a multispecific chimeric receptor comprising a polypeptide chain comprising: (a) an extracellular domain comprising a first NKG2D domain, a second NKG2D domain and a second antigen binding domain; (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the extracellular domain comprises, from N-terminus to C-terminus, a second antigen binding domain, a first NKG2D domain and a second NKG2D domain. In some embodiments, the second antigen binding domain is fused to the first NKG2D domain via a peptide linker. In some embodiments, the peptide linker is no more than about 50 amino acids in length. In some embodiments, the peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 12-15.

One aspect of the present application provides a multispecific chimeric receptor comprising a first polypeptide chain and a second polypeptide chain, each chain comprising: (a) an extracellular domain comprising an NKG2D domain and a second antigen Binding domain; (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, each extracellular domain further comprises a dimerization motif. In some embodiments, the dimerization motif is displaced between the NKG2D domain and the second antigen binding domain. In some embodiments, the dimerization motif is a leucine zipper or cysteine zipper. In some embodiments, the second antigen binding domain is fused to the NKG2D domain via a peptide linker. In some embodiments, the peptide linker is no more than about 50 amino acids in length. In some embodiments, the peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 12-15. In some embodiments, the NKG2D domain of the first polypeptide chain is crosslinked to the NKG2D domain of the second polypeptide chain through one or more disulfide bonds. In some embodiments, each of the first polypeptide chain and the second polypeptide chain comprises from N-terminus to C-terminus: a second antigen binding domain, an NKG2D domain, a transmembrane domain, and the intracellular signaling domain. .

In some embodiments according to any one of the multispecific chimeric receptors described above, the multispecific chimeric receptor is a bispecific chimeric receptor.

In some embodiments according to any one of the multispecific guimeric receptors described above, the second antigen binding domain is an antibody fragment. In certain embodiments, the antibody fragment is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, GD-2, NY- It specifically binds to an antigen selected from the group consisting of ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is a ligand or ligand binding domain. In some embodiments, the ligand or ligand binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the second antigen binding domain is an IL-3 domain. In some embodiments, the IL-3 domain is at least about 85% ( e.g. , at least about 90%, 92%, 95%, 98%) relative to the amino acid sequence of SEQ ID NO:9, or the amino acid sequence of SEQ ID NO:9. , Or 99%) variants thereof with sequence identity.

In some embodiments according to any one of the chimeric receptors or multispecific chimeric receptors described above, the NKG2D domain, or the first NKG2D domain and/or the second NKG2D domain, is the amino acid sequence of SEQ ID NO: 7 or 8, or SEQ ID NO: 7 or 8 with respect to the amino acid sequence of at least about 85% ( eg , at least about 90%, 92%, 95%, 98%, or 99%) sequence identity thereof.

In some embodiments according to any one of the chimeric receptors or multispecific chimeric receptors described above, the transmembrane domain is a molecule selected from the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152 and PD1. Is derived from In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 4 or 45.

In some embodiments according to any one of the chimeric receptors or multispecific chimeric receptors described above, the intracellular signaling domain comprises an intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 6.

In some embodiments according to any one of the chimeric receptors or multispecific chimeric receptors described above, the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, ICOS, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, the co-stimulatory signaling domain comprises a cytoplasmic domain of CD28 and/or a cytoplasmic domain of 4-1BB. In some embodiments, the co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 5.

In some embodiments according to any one of the chimeric receptors or multispecific chimeric receptors described above, the chimeric receptor or multispecific chimeric receptor is between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. It further includes a hinge area located at. In some embodiments, the hinge region is derived from CD8α. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 3.

In some embodiments, one or more isolated nucleic acid(s) is provided comprising a nucleic acid sequence encoding one or more of the polypeptide chains in any one of the chimeric receptors or multispecific chimeric receptors described above.

One aspect of the present application provides a dual chimeric receptor system comprising: (i) a first chimeric receptor comprising a first polypeptide chain and a second polypeptide chain, each chain comprising: (a) a second 1 extracellular domain comprising the NKG2D domain; (b) a first transmembrane domain; And (c) a first intracellular signaling domain; And (ii) a second chimeric receptor comprising a third polypeptide chain, wherein the chain comprises: (a) a second extracellular domain comprising a second antigen binding domain; And (b) a second transmembrane domain. In some embodiments, the second chimeric receptor further comprises a second intracellular signaling domain.

One aspect of the present application provides a dual chimeric receptor system comprising: (i) a first chimeric receptor comprising a first polypeptide chain, wherein the chain comprises: (a) a first NKG2D domain and a first 2 a first extracellular domain comprising the NKG2D domain; (b) a first transmembrane domain; And (c) a first intracellular signaling domain; And (ii) a second chimeric receptor comprising a second polypeptide chain, wherein the chain comprises: (a) a second extracellular domain comprising a second antigen binding domain; And (b) a second transmembrane domain. In some embodiments, the second chimeric receptor further comprises a second intracellular signaling domain.

In some embodiments according to any one of the dual chimeric receptor systems described above, the second antigen binding domain is an antibody fragment. In certain embodiments, the antibody fragment is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, GD-2, NY- It specifically binds to an antigen selected from the group consisting of ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is a ligand or ligand binding domain. In some embodiments, the ligand or ligand binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the second antigen binding domain is an IL-3 domain. In some embodiments, the IL-3 domain is at least about 85% ( e.g. , at least about 90%, 92%, 95%, 98%) relative to the amino acid sequence of SEQ ID NO:9, or the amino acid sequence of SEQ ID NO:9. , Or 99%) variants thereof with sequence identity.

In some embodiments according to any one of the dual chimeric receptor systems described above, the NKG2D domain, or the first NKG2D domain and/or the second NKG2D domain, is the amino acid sequence of SEQ ID NO: 7 or 8, or SEQ ID NO: 7 Or variants thereof having at least about 85% (eg, at least about 90%, 92%, 95%, 98%, or 99%) sequence identity to the amino acid sequence of 8.

In some embodiments according to any one of the dual chimeric receptor systems described above, the first and/or second transmembrane domain is the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152 and PD1. Is derived from a molecule selected from In some embodiments, the first and/or second transmembrane domain comprises the amino acid sequence of SEQ ID NO: 4 or 45.

In some embodiments according to any one of the dual chimeric receptor systems described above, the first and/or second intracellular signaling domain comprises an intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 6.

In some embodiments according to any one of the dual chimeric receptor systems described above, the first and/or second intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, the co-stimulatory signaling domain comprises a cytoplasmic domain of CD28 and/or a cytoplasmic domain of 4-1BB. In some embodiments, the co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 5.

In some embodiments according to any one of the dual chimeric receptor systems described above, the first and/or second chimeric receptor is a hinge located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. Include more areas. In some embodiments, the hinge region is derived from CD8α. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 3.

In some embodiments, one or more isolated nucleic acid(s) comprising nucleic acid sequences encoding one or more polypipted chains in any one of the dual chimeric receptor systems described above are provided. In some embodiments, an isolated nucleic acid is provided comprising a first nucleic acid sequence encoding a first chimeric receptor and a second nucleic acid sequence encoding a second chimeric receptor, wherein the first nucleic acid sequence is self-cleaving. -cleaving) is operably linked to a second nucleic acid sequence via a third nucleic acid sequence encoding the peptide. In some embodiments, the self-cleaving peptide is a T2A, P2A, or F2A peptide.

In some embodiments, at least about 85% (e.g., at least about 90%) relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-20 and SEQ ID NOs: 33-35, or SEQ ID NOs: 16-20 and 33-35. %, 92%, 95%, 98%, or 99%) a chimeric receptor comprising an amino acid sequence selected from the group consisting of variants thereof having sequence identity. In some embodiments, at least about 85% (e.g., at least about 90%, 92%, 95%, 98%, or 99%) sequence relative to the amino acid sequence of SEQ ID NO: 34, or the amino acid sequence of SEQ ID NO: 34. A first chimeric receptor comprising a variant thereof having identity; And a variant thereof having at least about 85% (e.g., at least about 90%, 92%, 95%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, or to the amino acid sequence of SEQ ID NO: 41. A dual chimeric receptor system comprising a second chimeric receptor comprising a is provided. In some embodiments, at least about 85% (e.g., at least about 90%, 92%, 95%, 98%, or 99%) sequence relative to the amino acid sequence of SEQ ID NO: 35, or the amino acid sequence of SEQ ID NO: 35. A first chimeric receptor comprising a variant thereof having identity; And a variant thereof having at least about 85% (e.g., at least about 90%, 92%, 95%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 42, or to the amino acid sequence of SEQ ID NO: 42. A dual chimeric receptor system comprising a second chimeric receptor comprising a is provided. In some embodiments, at least about 85% ( e.g. , at least about 90%, 92%, 95%, 98%, or with respect to the amino acid sequence of SEQ ID NO: 36 or 37, or the amino acid sequence of SEQ ID NO: 36 or 37 99%) A polypeptide comprising a variant thereof having sequence identity is provided. In some embodiments, at least about 85% (e.g., at least about 90%, 92) for a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 21-27 and 38-40, or SEQ ID NOs: 21-27 and 38-40. %, 95%, 98%, or 99%) an isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of variants thereof having sequence identity.

In some embodiments, one or more vectors encoding any one or more of the isolated nucleic acids described above are provided. In some embodiments, the vector is a lentiviral vector.

Another aspect of the present application provides an engineered immune effector cell comprising any one of the chimeric receptors, multispecific chimeric receptors or dual chimeric receptor systems described above, isolated nucleic acids, or vectors. In certain embodiments, the immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMC), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells. In some embodiments, the immune effector cell is a T cell.

In some embodiments, a pharmaceutical composition is provided comprising any one of the engineered immune effector cells described above, and a pharmaceutically acceptable carrier.

Another aspect of the present application provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of any one pharmaceutical composition described above. In certain embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia.

Also provided are methods of making and using kits and articles comprising any one of the chimeric receptors, multispecific chimeric receptors, dual chimeric receptor systems, engineered immune effector cells, isolated nucleic acids, or vectors described above. .

1A shows a schematic diagram of an exemplary bispecific chimeric receptor (LIC2001) comprising a single polypeptide chain comprising an IL-3 domain and two NKG2D domains. The positively charged residue (R) is engineered at the N-terminus ( e.g. , reverse NKG2D domain) of the first NKG2D domain, and the negatively charged residue (D) is the C-terminus ( e.g. , forward) of the second NKG2D domain. (forward) NKG2D domain). The engineered R residue and D residue form a salt bridge, and dimerization is promoted with each other.
1B shows a schematic diagram of an exemplary bispecific chimeric receptor (LIC2001-1) comprising a single polypeptide chain comprising an IL-3 domain and two NKG2D domains.
1C shows a schematic diagram of an exemplary bispecific chimeric receptor comprising two polypeptide chains, each chain comprising an IL-3 domain, a leucine zipper motif, and an NKG2D domain. The leucine zipper motif of each polypeptide chain promotes dimerization. Additionally, the NKG2D domains can be cross-linked with each other through disulfide bonds. LIC2002 includes an IL-3 domain, a leucine zipper motif, and a reverse NKG2D domain. LIC2002-2 contains an IL-3 domain, a leucine zipper motif, and a forward NKG2D domain.
1D shows a schematic diagram of an exemplary bispecific chimeric receptor (LIC2002-1) comprising two polypeptide chains, each chain comprising an IL-3 domain, a leucine zipper motif, and an NKG2D domain. The NKG2D domains are crosslinked with each other through disulfide bonds.
1E shows a schematic diagram of an exemplary dual chimeric receptor system (LIC2003) comprising a first chimeric receptor targeting an NKG2D ligand, and a second chimeric receptor targeting CD123. The first chimeric receptor comprises a polypeptide chain comprising two NKG2D domains, which two chains can be crosslinked to each other through disulfide bonds. The second chimeric receptor comprises an IL-3 domain. The second chimeric receptor may or may not contain an intracellular signaling domain.
1F shows a schematic diagram of an exemplary dual chimeric receptor system (LIC2004) comprising a first chimeric receptor targeting an NKG2D ligand, and a second chimeric receptor targeting CD123. The first chimeric receptor contains two polypeptide chains, each of which contains a single NKG2D domain, wherein the NKG2D domains are cross-linked with each other through disulfide bonds. The second chimeric receptor comprises an IL-3 domain. The second chimeric receptor may or may not contain an intracellular signaling domain. This figure shows an exemplary second IL-3 chimeric receptor that does not have an intracellular signaling domain.
Figure 2 shows the expression of NKG2D × IL-3 chimeric receptor constructs (LIC2004 and LIC2002-2) in engineered T cells as measured by flow cytometry.
3A-3C show the in vitro cytotoxic activity of engineered T cells expressing various NKG2D×IL-3 chimeric receptor constructs against tumor cells: K562-CD123-Luc (FIG. 3A ), K562-Luc (FIG. 3B ). ) And KG1-Luc (Figure 3c).
4 shows a plot comparing the dose-dependent cytotoxic activity of engineered T cells expressing various chimeric receptor constructs against K562-CD123-Luc.
Figures 5A-5B show the dose-dependent cytotoxic activity of engineered T cells expressing various chimeric receptor constructs against tumor cells: K562-CD123-Luc (Figure 5A) and K562-Luc (Figure 5B). "NKG2D-CD123 binder" refers to engineered T cells expressing the LIC2004 dual chimeric receptor system. “NKG2D” refers to engineered T cells that express only chimeric receptors containing the NKG2D domain in the LIC2004 dual chimeric receptor system ( eg , LIC2004-1). “CD123 conjugate” refers to engineered T cells that express only chimeric receptors containing only the IL-3 domain in the LIC2004 dual chimeric receptor system.
6A shows the blocking effect of MICA (a cognate ligand of NKG2D) by engineered T cells expressing the NKG2D × IL-3 chimeric receptor construct in the killing of K562-CD123-Luc and K562-Luc cells. Figure 6b shows that BSA does not have a significant blocking effect by engineered T cells expressing the NKG2D × IL-3 chimeric receptor construct in killing K562-CD123-Luc and K562-Luc cells.
7 shows the cytotoxic activity of engineered T cells expressing the LIC2004 construct and the LIC2002-2 construct against K562 and K562-CD123-Luc tumor cells.
Figures 8a-8c is by co-culturing engineered T cells expressing LIC2002-2, LIC2004 and LIC2004-1 constructs with K562-CD123-Luc, K562-Luc and KG1-Luc cell lines, secretion of IFNγ Show the level.

Detailed description of this application

This application provides an NKG2D ligand and a second antigen, CD123 For instance (e.g., IL-3 domains) multiple targeting a specific (e.g. bispecific) chimeric receptors and a chimeric dual receptor system. In some embodiments, unlike antibody-based CARs, the chimeric receptor and dual chimeric receptor systems described herein utilize a high affinity and specificity between a ligand and its cognate receptor on T cells. The NKG2D ligand is expressed only on stressed cells, especially tumor cells. Engineered immune cells expressing the NKG2D chimeric receptor and dual chimeric receptor systems described herein have enhanced anti-tumor capacity and provide useful therapeutic agents for anti-cancer therapy.

Thus, one aspect of the present application provides a multispecific chimeric receptor comprising: (a) an extracellular domain comprising an NKG2D domain and a second antigen binding domain ( eg , a binding domain targeting CD123); (b) transmembrane domain; And (c) an intracellular signaling domain.

In some embodiments, a multispecific chimeric receptor comprising a polypeptide chain is provided, wherein the chain comprises: (a) a second antigen binding domain ( eg , IL-3 domain), a first NKG2D domain And an extracellular domain comprising a second NKG2D domain; (b) a transmembrane domain, and (c) an intracellular signaling domain.

In some embodiments, a multispecific chimeric receptor is provided comprising a first polypeptide chain and a second polypeptide chain, each chain comprising: (a) an extracellular domain comprising an NKG2D domain and a second antigen. Binding domain ( eg , IL-3 domain); (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the extracellular domain further comprises a dimerization motif, such as a leucine zipper.

In another aspect, a dual chimeric receptor system is provided comprising: (i) a first chimeric receptor comprising: (a) an extracellular domain comprising a first NKG2D domain; (b) a first transmembrane domain; And (c) a first intracellular signaling domain; (ii) a second chimeric receptor comprising: (a) a second extracellular domain comprising a second antigen binding domain ( eg , an IL-3 domain); (b) a second transmembrane domain; And optionally (c) a second intracellular signaling domain. In some embodiments, the first chimeric receptor comprises a single polypeptide chain, wherein the first extracellular domain comprises a first NKG2D domain and a second NKG2D domain. In some embodiments, the first chimeric receptor comprises a first polypeptide chain and a second polypeptide chain, wherein each chain comprises: (a) an extracellular domain comprising a first NKG2D domain; (b) a first transmembrane domain; And (c) a first intracellular signaling domain.

Engineered immune effector cells (such as T cells) comprising the chimeric receptor, multispecific chimeric receptor or dual chimeric receptor system, pharmaceutical compositions, kits, articles of manufacture and cancer using the engineered immune effector cells Methods of doing are also described herein.

I. Definition

As used herein, “chimeric receptor” refers to a genetically engineered receptor, which is through antigen-antibody interactions or ligand-receptor binding, on one or more immune effector cells, such as T cells. It can be used to graft specific polypeptide interactions. Some chimeric receptors are also known as “chimeric antigen receptors”, “artificial T-cell receptors”, “chimeric T cell receptors”, or “chimeric immune receptors.” In some embodiments, the chimeric receptor is one or more antigenic receptors. An extracellular antigen-binding domain specific for (such as a tumor antigen), a transmembrane domain, and an intracellular signaling domain of T cells and/or other receptors In some embodiments, the extracellular antigen-binding domain is At least one domain derived from a ligand, or the extracellular domain of a receptor, wherein the ligand or receptor comprises a cell surface antigen, such as a tumor antigen.

“NKG2D chimeric receptor” refers to a chimeric receptor having an extracellular domain comprising one or more binding domains specific for an extracellular domain NKG2D ligand ( eg , NKG2D domain). “NKG2D × IL-3 chimeric receptor” refers to a chimeric receptor having an extracellular domain including a binding domain specific for NKG2D ligand ( eg , NKG2D domain) and a binding domain specific for CD123 ( eg , IL-3 domain). do.

As used herein, “NKG2D domain” refers to a functional fragment in the extracellular domain of the NKG2D, which is capable of specifically binding to one or more NKG2D ligands upon dimerization of the NKG2D domain. Exemplary human NKG2D ligands include, but are not limited to, MICA, MICB, and ULBP molecules.

As used herein, “IL-3 domain” refers to a functional fragment of IL-3 (including full-length IL-3), which is CD123, such as the IL-3R complex and/or the IL-3RA subunit ( subunit).

1 μM,

100 nM,

10 nM,

1 nM, 또는

0.1 nM의 해리상수(Kd)를 갖는다. 일부 구체예들에서, 항원 결합 단백질은 상이한 종의 단백질중에서 보존된 단백질 상에 있는 에피토프에 특이적으로 결합한다. 일부 구체예들에서, 특이적 결합은 독점적(exclusive) 결합을 포함할 수 있지만, 이것을 요구하지는 않는다.As used herein, the terms "target", "specifically bind", "specifically recognize", or "specific to" means a measurable and reproducible interaction, such as a target and Refers to the binding between antigen binding proteins (such as antigen binding domains, ligands, or chimeric receptors), which determines the presence of a target in the presence of a population of hybrid molecules, including biological molecules, for example specific to the target. An antigen binding protein that binds operatively is an antigen binding protein that binds to a target with greater affinity, avidity, more immediately, and/or for a longer period of time compared to binding to other targets. In examples, the degree of binding of the antigen-binding protein to an irrelevant target is less than about 10% of the binding of the antigen-binding protein to the target, as measured, for example , by radioimmunoassay (RIA). , The antigen-binding protein that specifically binds to the target

1 μM,

100 nM,

10 nM,

1 nM, or

It has a dissociation constant (Kd) of 0.1 nM. In some embodiments, the antigen binding protein specifically binds an epitope on a protein conserved among proteins of different species. In some embodiments, specific binding can include, but does not require, exclusive binding.

The term “specificity” refers to the selective recognition of an antigen binding protein (such as an antigen binding domain, ligand or chimeric receptor) for a specific epitope of an antigen. The term “multispecific” as used herein indicates that an antigen binding protein (such as a chimeric receptor) has two or more antigen-binding sites, of which at least two bind different antigens. “Bispecific” as used herein indicates that an antigen binding protein (such as a chimeric receptor) has two different antigen-binding specificities.

“Binding affinity” generally refers to the total strength of a non-covalent interaction between a molecule (such as an antigen binding domain, ligand, or chimeric receptor) and a single binding site of its binding partner ( such as an antigen). Unless stated otherwise, as used herein, “binding affinity” refers to an intrinsic binding affinity that reflects a 1:1 interaction between the components of a binding pair ( eg , an antigen binding domain and an antigen). The affinity of a molecule X for its partner Y can be expressed as the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high-affinity antibodies generally bind antigens faster and tend to bind longer. Various methods of measuring binding affinity are known in the art, any of which may be used for the purposes of this application.

The term “antibody” refers to a monoclonal antibody (including a full-length 4-chain antibody or a full-length heavy chain antibody having an immunoglobulin Fc region), multiple epitope specificity, multispecific antibody ( eg , bispecific antibody, diabody , And single-chain molecules), as well as antibody fragments ( eg , Fab, F(ab') ₂ , and Fv). Antibodies contemplated herein include single-domain antibodies, such as antibodies only with heavy chains.

“Antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding and/or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments; Diabodies; Linear antibodies (US Patent No. 5,641,870, Example 2; Zapata et al . , Protein Eng. 8(10): 1057-1062 [1995]); Single-chain antibody molecules; Single-domain antibodies (such as V _H H), and multispecific antibodies formed from antibody fragments.

Also abbreviated as “sFv” or “scFv”, “single-chain Fv” are antibody fragments comprising V _H and V _L antibody domains linked to a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the V _H domain and the V _L domain, which makes the sFv a desirable structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Mon℃lonal Antibodies , vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. See 269-315 (1994).

For peptide, polypeptide or antibody sequences, "% amino acid sequence identity" and "homology" align reference sequences and candidate sequences, and introduce gaps to obtain maximum percentage sequence identity as needed. Then, it is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, and any conservative substitutions are not considered part of sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art using publicly available computer software, for example BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. I can. One of skill in the art can determine the appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequence being compared.

An “isolated” nucleic acid molecule encoding a chimeric receptor or dual chimeric receptor system described herein is a nucleic acid molecule that has been identified and isolated from at least one contaminating nucleic acid molecule commonly associated in the environment in which it was created. Preferably, the isolated nucleic acid is not associated with all other components associated with its production environment. Isolated nucleic acid molecules encoding the polypeptides and antibodies described herein exist in a form that is not a form or set-up as they are found in nature. Thus, isolated nucleic acid molecules are distinct from nucleic acids encoding the polypeptides and antibodies described herein that exist naturally in cells.

The term “regulatory sequence” refers to a DNA sequence required for the expression of a coding sequence that is operably linked in a particular host organism. Regulatory sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals and enhancers.

A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to the DNA of the polypeptide when expressed as a preprotein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to the coding sequence when it affects the transcription of the sequence; Or the ribosome binding site is operably linked to the coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequence is contiguous, in the case of a secretory leader, that is contiguous and in the reading phase. However, enhancers need not be contiguous. Linking is accomplished by ligation at conventional restriction sites. If no such site is present, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid linked thereto. The term refers to a vector as a self-replicating nucleic acid structure, and included in the genome of the host cell into which it has been introduced. Including vectors Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked, such vectors are referred to herein as “expression vectors”.

As used herein, the term “autologous” refers to any substance from which any substance derived from an individual can be re-introduced into that same individual.

"Allogeneic" refers to grafts derived from different individuals of the same species.

The term “transfected” or “transformed” or “transduced”, as used herein, refers to the process by which exogenous nucleic acid is delivered or introduced into a host cell. A “transfected” or “transformed” or “transduced” cell refers to a transfected, transformed or transduced cell with an exogenous nucleic acid. Cells include the original target cell and its progeny.

As used herein, the expressions “cell”, “cell line” and “cell culture” are used interchangeably, and all such names include progeny. Thus, the terms "transfectant" and "transfected cell" include the primary target cell and cultures derived therefrom, regardless of the number of metastases. It should also be understood that all progeny due to intentional or unintended mutations may not be exactly the same in DNA content. Variant progeny that have been selected from the originally transformed cells and have the same function or biological activity are also included herein.

As used herein, “treatment” or “treating” is an approach to obtaining beneficial or desirable outcomes, including clinical outcomes, for purposes of the present invention, beneficial or desirable clinical outcomes. Includes, but is not limited to, one or more of the following: alleviation of one or more symptoms caused by the disease, reduction of the severity of the disease, stabilization of the disease ( e.g. , preventing or delaying exacerbation of the disease), Preventing or delaying the spread ( e.g. , metastasis) of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, (partially or entirely) providing remission of the disease, Reducing the dose of one or more drugs required for treatment, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival, “treatment” also encompasses reducing the pathological consequences of cancer. The methods of the present application contemplate any of these one or more aspects of treatment.

As used herein, “individual” or “subject” refers to a mammal, including, but not limited to, humans, cattle, horses, cats, dogs, rodents, or primates. In some embodiments, the subject is a human.

As used herein, the term “effective amount” refers to the treatment of a specified disorder, condition or condition, such as an agent sufficient to ameliorate, alleviate, alleviate and/or delay one or more symptoms, such as engineered immunity. It refers to the amount of effector cells, or pharmaceutical compositions thereof. In the context of cancer, an effective amount is an amount sufficient for the tumor to contract and/or reduce the rate of growth of the tumor (e.g., inhibit tumor growth), or to prevent or delay other undesired cell proliferation. Includes. In some embodiments, the effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. Effective amounts may be administered in one or more administrations. The effective amount of the drug or composition is to (i) reduce the number of cancer cells; (ii) reducing tumor size; (iii) inhibiting, delaying, slowing, or preferably stopping cancer cell infiltration into surrounding organs to some extent; (iv) inhibit ( ie , delay to some extent, preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) preventing or delaying the occurrence and/or recurrence of tumors; And/or (vii) one or more symptoms associated with cancer may be eliminated to some extent.

As used herein, "delaying" the development of cancer means delaying, hindering, slowing, retarding, stabilizing and/or delaying the development of a disease. This delay can be of varying duration depending on the history of the disease and/or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can include prophylaxis in that the individual does not develop the disease in practice. A method of “delaying” the development of cancer is a method of reducing the probability of developing disease in a given time frame and/or reducing the degree of disease in a given time frame as compared to not using the method. These comparisons are generally based on clinical studies using a statistically significant number of subjects. Cancer development can be detected using standard methods including, but not limited to, computed tomography (CAT Scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation, arterial angiography or biopsy. Development may initially be undetectable and may refer to the progression of cancer, including onset, recurrence and onset.

It is understood that the embodiments of the present application described herein include “consisting” and/or “consisting essentially of” as embodiments.

Reference to "about" of a value or parameter in this specification includes (and describes) variations with respect to the value or parameter itself. For example, a description of “about X” includes a description of “X”.

As used herein, "not" a value or parameter generally means and describes "other than" a value or parameter. For example, that the method is not used to treat type X cancer means that the method uses cancer treatment other than type X.

As used herein, the term “about X-Y” has the same meaning as “about X to about Y”.

As used in the specification and claims, it should be noted that the singular ("a", "an" and "the") forms include the plural concept unless explicitly stated otherwise.

II. Multispecific chimeric receptor and dual chimeric receptor system

The present application provides a chimeric receptor and a chimeric receptor system targeting the NKG2D ligand. One aspect of the present application is an extracellular domain comprising an NKG2D domain and a second antigen binding domain, such as a CD123 binding domain, such as IL-3. It provides a multispecific chimeric receptor comprising a domain.

In some embodiments, a chimeric receptor is provided comprising: (a) an extracellular domain comprising an NKG2D domain; (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, the chimeric receptor further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the chimeric receptor comprises, in the N-terminal to C-terminal direction, the following: a first NKG2D domain, a peptide linker, a second NKG2D domain, a transmembrane domain (CD8α), derived from 4-1BB Co-stimulatory domain, and a primary signaling domain derived from CD3ζ. In some embodiments, the NKG2D domain comprises the amino acid sequence of SEQ ID NO: 8.

In some embodiments, a chimeric receptor is provided comprising a first polypeptide chain and a second polypeptide chain, each chain comprising: (a) an extracellular domain comprising an NKG2D domain and a dimerization motif ( e.g. , Leucine zippers or cysteine zippers); (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is crosslinked to the NKG2D domain of the second polypeptide chain through one or more disulfide bonds. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, each polypeptide chain further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain.

In some embodiments, a chimeric receptor is provided comprising: (a) an extracellular domain comprising a first NKG2D domain and a second NKG2D domain; (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, the chimeric receptor further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the chimeric receptor comprises, in the N-terminal to C-terminal direction, the following: a first NKG2D domain, a peptide linker, a second NKG2D domain, a transmembrane domain (CD8α), derived from 4-1BB Co-stimulatory domain, and a primary signaling domain derived from CD3ζ. In some embodiments, the NKG2D domain comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% for SEQ ID NO: 33 , 98%, 99%, or 100% sequence identity, comprising an amino acid sequence having at least about any one of. In some embodiments, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% relative to the nucleic acid sequence of SEQ ID NO: 38 An isolated nucleic acid sequence comprising a nucleic acid sequence having at least about any one of, 97%, 98%, 99%, or 100% sequence identity is provided.

In some embodiments, a multispecific ( e.g. , bispecific) chimeric receptor is provided comprising: (a) an extracellular domain comprising an NKG2D domain and a second antigen binding domain ( e.g. , IL-3 domain) ); (b) transmembrane domain; And (c) an intracellular signaling domain. In certain embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, It specifically binds to an antigen selected from the group consisting of GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, the multispecific chimeric receptor further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain.

The chimeric receptor ( eg , multispecific chimeric receptor) may comprise one or more polypeptide chains. In some embodiments, the chimeric receptor is a monomer. In some embodiments, the monomeric chimeric receptor comprises an extracellular domain comprising a first NKG2D domain and a second NKG2D domain. In some embodiments, the extracellular domain comprises, in the N-terminal to C-terminal direction, a second antigen binding domain ( eg , IL-3 domain), a first NKG2D domain and a second NKG2D domain. In some embodiments, the extracellular domain comprises, in the N-terminal to C-terminal direction, a first NKG2D domain, a second NKG2D domain, and a second antigen binding domain ( eg , IL-3 domain). . In some embodiments, the first NKG2D domain is crosslinked to the second NKG2D domain. In some embodiments, the first NKG2D domain comprises a first engineered residue at the N-terminus, and the second NKG2D domain comprises a second engineered residue at the C-terminus, wherein the first engineered residue is, for example, , Is associated with the second engineered moiety through a disulfide bond or a salt bridge. In some embodiments, the second antigen binding domain is via a peptide linker, such as a peptide linker of less than about 50 amino acids in length, such as a peptide linker comprising an amino acid sequence selected from SEQ ID NOs: 12-15. Fused to the NKG2D domain.

In some embodiments, the chimeric receptor ( eg , multispecific chimeric receptor) is a dimer, such as a homodimer or heterodimer. In some embodiments, the dimer chimeric receptor comprises two polypeptide chains, each chain comprising a single NKG2D domain. In some embodiments, the dimer chimeric receptor comprises two identical polypeptide chains. In some embodiments, the dimer chimeric receptor comprises two different polypeptide chains. In some embodiments, each polypeptide chain, from N-terminus to C-terminus, comprises: a second antigen binding domain, an NKG2D domain, a transmembrane domain, and the intracellular signaling domain. In some embodiments, each polypeptide chain, from N-terminus to C-terminus, comprises: an NKG2D domain, a second antigen binding domain, a transmembrane domain, and the intracellular signaling domain. In some embodiments, the NKG2D domains of each polypeptide chain of a dimer chimeric receptor are non-covalently associated with each other to form a dimer. In some embodiments, the NKG2D domains of each polypeptide chain of a dimeric chimeric receptor are covalently associated with each other, such as via a disulfide bond, and/or a dimerization motif in the extracellular domain ( e.g. , a leucine zipper, Or cysteine zipper) to form a dimer. In some embodiments, the second antigen binding domain is via a peptide linker, such as a peptide linker of less than about 50 amino acids in length, such as a peptide linker comprising an amino acid sequence selected from SEQ ID NOs: 12-15. Fused to the domain. In some embodiments, the second antigen binding domain is fused to the NKG2D domain through a dimerization motif, such as a leucine zipper or cysteine zipper.

Thus, in some embodiments, a multispecific ( e.g. , bispecific) chimeric receptor comprising a polypeptide chain is provided, wherein the chain comprises: (a) a first NKG2D domain, a second NKG2D domain , And an extracellular domain comprising a second antigen binding domain; (b) transmembrane domain; And (c) an intracellular signaling domain. In certain embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, It specifically binds to an antigen selected from the group consisting of GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the second antigen binding domain is via a peptide linker, such as a peptide linker of less than about 50 amino acids in length, such as a peptide linker comprising an amino acid sequence selected from SEQ ID NOs: 12-15. Fused to the NKG2D domain or the second NKG2D domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, the multispecific chimeric receptor further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the multispecific chimeric receptor further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of the polypeptide chain. In some embodiments, the polypeptide chain in the N-terminal to C-terminal direction comprises: a second antigen binding domain ( e.g. , IL-3 domain), a first peptide linker, a first NKG2D domain, 2 peptide linkers, a second NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain, a co-stimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ.

In some embodiments, a chimeric receptor is provided comprising a multispecific ( e.g. , bispecific) first polypeptide chain and a second polypeptide chain, each chain comprising: (a) comprising an NKG2D domain An extracellular domain and a second antigen binding domain; (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is crosslinked to the NKG2D domain of the second polypeptide chain through one or more disulfide bonds. In certain embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, It specifically binds to an antigen selected from the group consisting of GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the second antigen binding domain is via a peptide linker, such as a peptide linker of less than about 50 amino acids in length, such as a peptide linker comprising an amino acid sequence selected from SEQ ID NOs: 12-15. Fused to the domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, each polypeptide chain further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain in the N-terminal to C-terminal direction comprises: a second antigen binding domain ( eg , IL-3 domain), a peptide linker, NKG2D Domain, CD8α hinge region, CD8α transmembrane domain, co-stimulatory signaling domain derived from 4-1BB, and primary intracellular signaling domain derived from CD3ζ.

In some embodiments, a multispecific ( e.g. , bispecific) chimeric receptor comprising a first polypeptide chain and a second polypeptide chain is provided, each chain comprising: (a) comprising an NKG2D domain An extracellular domain, a dimerization motif ( eg , a leucine zipper or cysteine zipper), and a second antigen binding domain; (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is crosslinked to the NKG2D domain of the second polypeptide chain through one or more disulfide bonds. In certain embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, It specifically binds to an antigen selected from the group consisting of GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, each polypeptide chain further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first and second polypeptide chains in the N-terminal to C-terminal direction comprises: a second antigen binding domain ( eg , IL-3 domain), a leucine zipper, NKG2D Domain, CD8α hinge region, CD8α transmembrane domain, co-stimulatory signaling domain derived from 4-1BB, and primary intracellular signaling domain derived from CD3ζ.

In some embodiments, the second antigen binding domain specifically binds to a cell surface antigen, such as a tumor antigen. Exemplary tumor antigens are CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77, but is not limited thereto. In some embodiments, the second antigen binding domain is an antibody fragment, such as a single-chain antibody ( eg , scFv) or a single-domain antibody ( eg , VHH). In some embodiments, the second antigen binding domain is an antibody fragment ( eg, scFv or VHH) that specifically binds CD123 ( eg , IL-3R or IL-3RA subunit).

In some embodiments, the second antigen binding domain is a ligand. In some embodiments, the second antigen binding domain is a ligand-binding domain, such as an extracellular domain of a receptor. Exemplary ligands and receptors include, but are not limited to, NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the second antigen binding domain is an IL-3 domain.

Thus, in some embodiments, a multispecific (e.g., bispecific) chimeric receptor comprising a polypeptide chain is provided, wherein the chain comprises: (a) a first NKG2D domain, a second NKG2D domain , And an extracellular domain comprising a CD123 binding domain (eg, an IL-3 domain); (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the CD123 binding domain is the first NKG2D via a peptide linker, such as a peptide linker of less than about 50 amino acids in length, such as a peptide linker comprising an amino acid sequence selected from SEQ ID NOs: 12-15. Domain or a second NKG2D domain. In some embodiments, the CD123 binding domain is an anti-CD123 antibody fragment ( eg , scFv or VHH). In some embodiments, the CD123 binding domain is an IL-3 domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, the multispecific chimeric receptor further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the multispecific chimeric receptor further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of the polypeptide chain. In some embodiments, the polypeptide chain in the N-terminal to C-terminal direction, comprises: an IL-3 domain, a first peptide linker, a first NKG2D domain, a second peptide linker, a second NKG2D domain, CD8α hinge region, CD8α transmembrane domain, co-stimulatory signaling domain derived from 4-1BB, and primary intracellular signaling domain derived from CD3ζ. Exemplary multispecific chimeric receptors are shown in Figures 1A-1B.

In some embodiments, a multispecific (e.g., bispecific) chimeric receptor comprising a first polypeptide chain and a second polypeptide chain is provided, each chain comprising: (a) comprising an NKG2D domain An extracellular domain and a CD123 binding domain ( eg , IL-3 domain); (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is crosslinked to the NKG2D domain of the second polypeptide chain through one or more disulfide bonds. In some embodiments, the CD123 binding domain is via a peptide linker, such as a peptide linker of less than about 50 amino acids in length, such as a peptide linker comprising an amino acid sequence selected from SEQ ID NOs: 12-15. Is fused to In some embodiments, the CD123 binding domain is an anti-CD123 antibody fragment ( eg , scFv or VHH). In some embodiments, the CD123 binding domain is an IL-3 domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, each polypeptide chain further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain in the N-terminal to C-terminal direction comprises: an IL-3 domain, a peptide linker, an NKG2D domain, a CD8α hinge region, a CD8α transmembrane. Domain, a co-stimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ. An exemplary multispecific chimeric receptor is shown in Figure 1D.

In some embodiments, a multispecific (e.g., bispecific) chimeric receptor comprising a first polypeptide chain and a second polypeptide chain is provided, wherein each chain comprises: (a) comprises an NKG2D domain Extracellular domains, dimerization motifs ( eg , a leucine zipper, or cysteine zipper), and a CD123 binding domain ( eg , an IL-3 domain); (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is crosslinked to the NKG2D domain of the second polypeptide chain through one or more disulfide bonds. In some embodiments, the CD123 binding domain is an anti-CD123 antibody fragment ( eg , scFv or VHH). In some embodiments, the CD123 binding domain is an IL-3 domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, each polypeptide chain further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain in an N-terminal to C-terminal direction comprises: an IL-3 domain, a leucine zipper, a NKG2D domain, a CD8α hinge region, a CD8α transmembrane. Domain, a co-stimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ. An exemplary multispecific chimeric receptor is shown in Figure 1C.

Exemplary NKG2D x IL-3 chimeric receptors and their sequences are shown in Table 1. In the case of a dimer chimeric receptor having two identical polypeptide chains, the amino acid sequence of the monomer subunit is shown. In some embodiments, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% for an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-20. , 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. NKG2D × IL-3 chimeric receptor comprising a polypeptide having at least about any one of. In some embodiments, an NKG2D x IL-3 chimeric receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-20 is provided. In some embodiments, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% relative to the amino acid sequence of SEQ ID NO: 33. , 97%, 98%, 99%, or 100% sequence identity. NKG2D chimeric receptors comprising a polypeptide having at least about any one of. In some embodiments, an NKG2D chimeric receptor comprising the amino acid sequence of SEQ ID NO: 33 is provided. Polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-20 and 33 are also provided.

In some embodiments, one or more isolated nucleic acids encoding any one of a chimeric receptor or multispecific chimeric receptor provided herein are provided. In certain embodiments, wherein the chimeric receptor is a dimeric chimeric receptor having two identical polypeptide chains, a monomeric subunit of the chimeric receptor, such as an isolated nucleic acid encoding a single copy of the polypeptide chain is provided. In some embodiments, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% for a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 21-25 and 38. , 94%, 95%, 96%, 97%, 98%, 99%, or 100% of at least about any one of the isolated nucleic acids are provided. In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the isolated nucleic acid is RNA ( eg , mRNA). In some embodiments, one or more vectors comprising any one of the chimeric receptors described above or nucleic acids encoding multispecific chimeric receptors are provided. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector, such as a lentiviral vector. In some embodiments, the vector is a non-viral vector.

[Table 1]

Dual chimeric receptor system

One aspect of the present application provides a dual chimeric receptor system comprising: (i) a first chimeric receptor comprising: (a) an extracellular domain comprising a first NKG2D domain, (b) a first membrane A transverse domain, and (c) a first intracellular signaling domain; And (ii) a second chimeric receptor comprising: (a) a second extracellular domain comprising a second antigen binding domain, (b) a second transmembrane domain, and optionally (c) a second intracellular Signaling domain. The first chimeric receptor specifically binds to the NKG2D ligand and the second chimeric receptor specifically binds to a second antigen, such as a tumor antigen such as CD123.

Each of the first chimeric receptor and the second chimeric receptor may comprise one or more polypeptide chains. The dual chimeric receptor system may comprise any combination of a first chimeric receptor and a second chimeric receptor described herein.

In some embodiments, the first chimeric receptor comprises a single polypeptide chain comprising: (a) a first extracellular domain comprising a first NKG2D domain and a second NKG2D domain, (b) a first transmembrane Domain, and (c) a first intracellular signaling domain. In some embodiments, the first NKG2D domain is crosslinked to the second NKG2D domain. In some embodiments, the first NKG2D domain comprises a first engineered residue at the N-terminus, and the second NKG2D domain comprises a second engineered residue at the C-terminus, wherein the first engineered residue is, for example, , Is associated with the second engineered moiety through a disulfide bond or a salt bridge. In some embodiments, the first transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the first intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the first intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, the first chimeric receptor further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. In some embodiments, the first chimeric receptor further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of the polypeptide chain. In some embodiments, the first chimeric receptor comprises a polypeptide chain in the N-terminal to C-terminal direction, comprising: a first NKG2D domain, a peptide linker, a second NKG2D domain, a CD8α hinge region, CD8α. The transmembrane domain, a co-stimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ. An exemplary first chimeric receptor is shown in Figure 1E.

In some embodiments, the first chimeric receptor comprises a first polypeptide chain and a second polypeptide chain, wherein each chain comprises: (a) an extracellular domain comprising a first NKG2D domain, (b) A first transmembrane domain, and (c) a first intracellular signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is crosslinked to the NKG2D domain of the second polypeptide chain through one or more disulfide bonds. In some embodiments, the first transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the first intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the first intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, each polypeptide chain further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain in the N-terminus to C-terminus direction comprises: a NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain, derived from 4-1BB. A co-stimulatory signaling domain, and a primary intracellular signaling domain derived from CD3ζ. An exemplary first chimeric receptor is shown in FIG. 1F.

In some embodiments, the first chimeric receptor comprises a first polypeptide chain and a second polypeptide chain, wherein each chain comprises: (a) an extracellular domain comprising a first NKG2D domain and a dimerization domain. ( Eg, a leucine zipper, or a cysteine zipper), (b) a first transmembrane domain, and (c) a first intracellular signaling domain. In some embodiments, the dimerization domain is located at the N-terminus of the NKG2D domain. In some embodiments, the dimerization domain is located at the C-terminus of the NKG2D domain. In some embodiments, the NKG2D domain of the first polypeptide chain is further crosslinked to the NKG2D domain of the second polypeptide chain through one or more disulfide bonds. In some embodiments, the first transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the first intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the first intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, each polypeptide chain further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first and second polypeptide chains in the N-terminal to C-terminal direction comprises: a leucine zipper, a NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain, 4-1BB. A co-stimulatory signaling domain derived from and a primary intracellular signaling domain derived from CD3ζ.

In some embodiments, the second chimeric receptor comprises a polypeptide chain comprising: (a) a second extracellular domain comprising a second antigen binding domain, and (b) a second transmembrane domain. In some embodiments, the second chimeric receptor does not comprise an intracellular signaling domain. In some embodiments, the second extracellular domain further comprises an NKG2D domain, e.g., the second extracellular domain in the N-terminal to C-terminal direction, comprising: NKG2D domain and a second antigen Binding domain, or a second antigen binding domain and an NKG2D domain. In some embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, It specifically binds to an antigen selected from the group consisting of GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the second antigen binding domain is a CD123 binding domain, such as an IL-3 domain. In some embodiments, the second transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the second chimeric receptor further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the second extracellular domain and the N-terminus of the second transmembrane domain. In some embodiments, the second chimeric receptor further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of the polypeptide chain. In some embodiments, the second chimeric receptor comprises, in the N-terminal to C-terminal direction, a polypeptide comprising: an IL-3 domain, a CD8α hinge region, and a CD8α transmembrane domain. An exemplary second chimeric receptor is shown in FIGS. 1E and 1F.

In some embodiments, the second chimeric receptor comprises a polypeptide chain comprising: (a) a second extracellular domain comprising a second antigen binding domain, (b) a second transmembrane domain, and (c ) A second intracellular signaling domain. In some embodiments, the second extracellular domain further comprises an NKG2D domain, e.g., the second extracellular domain in the N-terminal to C-terminal direction, comprising: NKG2D domain and a second antigen Binding domain, or a second antigen binding domain and an NKG2D domain. In some embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, It specifically binds to an antigen selected from the group consisting of GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the second antigen binding domain is a CD123 binding domain, such as an IL-3 domain. In some embodiments, the second transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the second intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, the second intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the second intracellular signaling domain does not comprise a primary intracellular signaling domain. In some embodiments, the second chimeric receptor further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the second chimeric receptor further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of the polypeptide chain. In some embodiments, the second chimeric receptor comprises, in the N-terminal to C-terminal direction, a polypeptide comprising: an IL-3 domain, a CD8α hinge region, a CD8α transmembrane domain, and derived from 4-1BB. Co-stimulatory signaling domain.

In some embodiments, a dual chimeric receptor system is provided comprising: (i) each chain of a first chimeric receptor comprising a first polypeptide chain and a second polypeptide chain comprises: (a) a first An extracellular domain comprising an NKG2D domain, (b) a first transmembrane domain, and (c) a first intracellular signaling domain; And (ii) a second chimeric receptor comprising a third polypeptide chain comprising: (a) a second extracellular domain comprising a second antigen binding domain ( eg , an IL-3 domain), (b) a second A transmembrane domain, and optionally (c) a second intracellular signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is crosslinked to the NKG2D domain of the second polypeptide chain through one or more disulfide bonds. In some embodiments, the first transmembrane domain and/or the second transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the first intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the first intracellular signaling domain and/or the second intracellular domain comprises a co-stimulatory signaling domain. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, the second chimeric receptor does not comprise an intracellular signaling domain. In some embodiments, each polypeptide chain further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain in the N-terminus to C-terminus direction comprises: NKG2D domain, CD8α hinge region, CD8α transmembrane domain, derived from 4-1BB. A co-stimulatory signaling domain, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the third polypeptide chain, in the N-terminal to C-terminal direction, comprises an IL-3 domain and a CD8α transmembrane domain. An exemplary dual chimeric receptor system is shown in Figure 1F.

In some embodiments, the polypeptide(s) of the first chimeric receptor and the polypeptide(s) of the second chimeric receptor are expressed through a polycistronic nucleic acid construct. For example, the polypeptide(s) of a first chimeric receptor are fused to the polypeptide(s) of a second chimeric receptor via a self-cleaving peptide. Exemplary self-cleaving peptides include, but are not limited to, T2A, P2A, and F2A. The T2A peptide is, for example, Szymczak AL. Et al., Correction of multi-gene deficiency in vivo using a “self-cleaving” 2A peptide-based retroviral vector. Nat Biotechnol 2004; See 22(5)589-594.

In some embodiments, one or more isolated nucleic acids encoding any one of the dual chimeric receptor systems described herein are provided. In some embodiments, an isolated nucleic acid is provided comprising a first nucleic acid sequence encoding a first chimeric receptor and a second nucleic acid sequence encoding a second chimeric receptor, wherein the first nucleic acid sequence is a self-cleaving peptide ( For example , it is operably linked to a second nucleic acid sequence via a third nucleic acid sequence encoding T2A). In some embodiments, the first chimeric receptor is a dimeric chimeric receptor having two identical polypeptide chains, and the first nucleic acid encodes a monomer subunit of the first chimeric receptor, such as a single copy of the polypeptide chain. .

In some embodiments, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 for an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-35 and 41-42. Chimeric receptors are provided comprising an amino acid sequence having at least about any one of %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, an NKG2D × IL-3 dual chimeric receptor system is provided, which is 85%, 86%, 87%, 88%, 89%, 90%, 91%, relative to the amino acid sequence of SEQ ID NO: 34, A first chimeric receptor comprising an amino acid sequence having at least about any one of 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and SEQ ID NO: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% for the amino acid sequence of 41 , Or a second chimeric receptor comprising an amino acid sequence having at least about any one of 100% sequence identity. In some embodiments, an NKG2D x IL-3 dual chimeric receptor system is provided, which is 85%, 86%, 87%, 88%, 89%, 90%, 91%, relative to the amino acid sequence of SEQ ID NO: 35, A first chimeric receptor comprising an amino acid sequence having at least about any one of 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and SEQ ID NO: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% for the amino acid sequence of 42 , Or a second chimeric receptor comprising an amino acid sequence having at least about any one of 100% sequence identity. In some embodiments, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, relative to SEQ ID NO: 36 or 37, Polypeptides comprising an amino acid sequence having at least about any one of 97%, 98%, 99%, or 100% sequence identity are provided. In some embodiments, 85%, 86%, 87%, 88%, 89%, 90%, 91% for a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 26-27, 39-40 and 43-44. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of at least about any one of the isolated nucleic acids are provided. In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the isolated nucleic acid is RNA ( eg , mRNA). In some embodiments, one or more vectors comprising any of the dual chimeric receptor systems described above or one or more nucleic acids encoding chimeric receptors are provided. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector, such as a lentiviral vector. In some embodiments, the vector is a non-viral vector. An exemplary dual chimeric receptor system is shown below:

[Table 2]

Extracellular domain

The chimeric receptor, including the multispecific chimeric receptor, a first chimeric receptor and a second chimeric receptor of the dual chimeric receptor system described herein, comprises one or more NKG2D domains and/or a second antigen binding domain. Or more (such as any one of 1, 2, 3, 4, 5, 6 or more) antigen binding domains. The NKG2D domain and the second antigen binding domain can be fused directly to each other through a peptide bond, through a peptide linker, or through a dimerization motif, such as a leucine zipper or a cysteine zipper.

NKG2D domain

The extracellular domain of the chimeric receptor contains one or more NKG2D domains. In some embodiments, the extracellular domain of the chimeric receptor comprises a single NKG2D domain. In some embodiments, the extracellular domain comprises a first NKG2D domain and a second NKG2D domain. In some embodiments, the first NKG2D domain and the second NKG2D domain are the same. In some embodiments, the first NKG2D domain and the second NKG2D domain are different. In some embodiments, the first NKG2D domain and the second NKG2D domain may be directly fused to each other through a peptide bond or through a peptide linker.

In some embodiments, the NKG2D domain is derived from a human NKG2D molecule. In some embodiments, the NKG2D domain is derived from the extracellular domain of the NKG2D, such as human NKG2D. In some embodiments, the NKG2D domain is a domain having an amino acid sequence in the same order as a forward NKG2D domain, such as a wild type NKG2D domain. In some embodiments, the NKG2D domain comprises any one of at least about 100, 105, 110, 115, 120, 125, 130, 135, 140, 150 or more amino acids from the extracellular domain of wild-type NKG2D. In some embodiments, the NKG2D domain is a reverse NKG2D domain, such as a domain having a reverse sequence of amino acid sequence of a wild type NKG2D domain. In some embodiments, the NKG2D domain (including the first NKG2D domain and/or the second NKG2D domain) is 85%, 86%, 87%, 88%, 89%, 90%, 91 relative to SEQ ID NO: 7 or 8. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with at least about any one of the amino acid sequences. In some embodiments, the NKG2D domain comprises at least 1, 2, 3, 4, 5 or more amino acid substitutions ( eg , conserved amino acid substitutions) when compared to the corresponding wild-type sequence of NKG2D.

NKG2D is a unique member of the NKG2 family, a C-type lectin receptor that stimulates or inhibits the cytotoxic activity of NK cells. NKG2D is a type II transmembrane-immobilized glycoprotein that is primarily expressed on the surface of NK cells and CD8 ⁺ T cells ( eg , αβ T cells and γδ T cells). 70% sequence identity is shared between the human and murine receptors, and is highly conserved across species. Unlike other NKG2 receptors that heterodimerize to CD94 and bind to nonclassical MHC glycoprotein class I, NKG2D forms homodimers and binds to cellular stress-inducing molecules. Accumulating evidence indicates that NKG2D plays an important role in immunosurveillance against stressed or abnormal cells such as autologous tumor cells and virus-infected cells.

Various NKG2D ligands have been identified in humans, including MIC molecules (MHC class I chain-associated protein A and protein B) encoded by genes within the MHC family clustered on human chromosome 6 (Bahram et al. 2005). , Or MICA and MICB), and ULBP molecules (UL16-binding protein, also known as RAET1 protein). All NKG2D ligands are homologous to the MHC class I molecule and show significant allelic variation. Although NKG2D ligand RNAs are widely expressed in all tissues and organs of the body, NKG2D ligands are generally absent from the normal adult cell surface (Le Bert and Gasser 2014). However, expression of NKG2D ligands is induced or upregulated primarily in tissues of epithelial origin, in response to cellular stresses including heat shock, DNA damage and halted DNA replication. The presence of the NKG2D ligand on the cell flags the cell's activity for NK cell targeting and potential elimination (Le Bert and Gasser 2014). Interestingly, the high activity of the DNA repair pathway in transformed cells across a variety of hematologic and solid tumors induces the expression of the NKG2D ligand, which makes these cells susceptible to NK-mediated lysis (Sentman et al. 2006). ).

NKG2D is encoded by the KLRK1 gene. NKG2D is a transmembrane receptor protein comprising the following three domains: a cytoplasmic domain (residues 1-51 of human NKG2D), a transmembrane domain (residues 52-72 of human NKG2D), and an extracellular domain (residues 73 of human NKG2D). -216). The extracellular domain of NKG2D contains a C-type lectin domain (residues 98-213 of human NKG2D).

Second antigen binding domain

The second antigen binding domain specifically binds to the cell surface molecule. The second antigen binding domain can be selected to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. Antigens targeted by the second antigen binding domain can be directly or indirectly involved in this disease. In some embodiments, the antigen is a tumor antigen. In some embodiments, the tumor antigen is associated with B cell malignancies.

Tumor antigens are proteins produced by tumor cells capable of eliciting an immune response, especially a T-cell mediated immune response. The selection of the targeted antigen of the invention will depend on the specific type of cancer being treated. Exemplary tumor antigens include, for example, glioma-associated antigen, cancer embryonic antigen (CEA), β-human chorionic gonadotropin, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE- 1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), visceral carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prosteine, PSMA, HER2/neu, sulvibin and telomerase, prostate-epithelial cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, Ho Neutral elastase, epirin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSA is specific to tumor cells and does not occur in other cells in the body. TAA-associated antigens are not specific to tumor cells, but instead are expressed in normal cells under conditions that do not induce immunological resistance to the antigen. Expression of this antigen in the tumor can occur under conditions in which the immune system can respond to the antigen. TAAs can be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, and can be present at very low levels normally in normal cells, but at much higher levels in tumors. I can. It.

Non-limiting examples of TSA or TAA antigens are as follows: differentiation antigens such as MART-1/MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor- Specific multilineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; Overexpressed embryonic antigens such as CEA; Overexpressed oncogenes and mutated tumor-suppressant genes, such as p53, Ras, HER2/neu; Unique tumor antigens due to chromosomal translocations (transl°Cations); Such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; And viral antigens such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta- HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp -175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein \cyclophyllin C-associated protein, TAAL6, TAG72, TLP, and TPS.

The second antigen binding domain can be in any suitable format. In some embodiments, the second antigen binding domain is derived from an antibody, such as a 4-chain antibody, or a single-domain antibody, such as only a heavy chain antibody. In some embodiments, the second antigen binding domain is an antibody fragment, such as Fab, Fv, scFv, or VHH. In certain embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, GD-2, It is an antibody fragment that specifically binds to an antigen selected from the group consisting of NY-ESO-1, MAGE A3, and glycolipid F77.

In some embodiments, the second antigen binding domain is a ligand of a receptor, or a ligand binding domain. In some embodiments, the second antigen binding domain is a ligand or ligand binding domain derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. to be.

CD123-binding domain

In some embodiments, the second antigen binding domain is a CD123-binding domain. In some embodiments, the CD123-binding domain is an antibody fragment ( eg , scFv or VHH) of an anti-CD123 antibody. In some embodiments, the CD123-binding domain is a ligand of CD123, or an IL-3 domain. In some embodiments, the IL-3 domain is derived from human IL-3, such as full length human IL-3 or a functional fragment thereof. In certain embodiments, the CD123-binding domain is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% relative to SEQ ID NO:9. , 96%, 97%, 98%, 99%, or 100% sequence identity with at least about any one of the amino acid sequences.

In some embodiments, a chimeric receptor is provided comprising: (a) an extracellular domain comprising a CD123-binding domain; And (b) a transmembrane domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the chimeric receptor further comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain does not comprise the primary intracellular signaling domain of an immune effector cell. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain consists of (or consists essentially of) one or more co-stimulatory signaling domains. In certain embodiments, the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. It is derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. In some embodiments, the chimeric receptor further comprises a hinge region (such as a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the chimeric receptor comprises, in the N-terminal to C-terminal direction, a CD123-binding domain and a transmembrane domain (CD8α). In some embodiments, the CD123-binding domain is an IL-3 domain. In some embodiments, the CD123-binding domain comprises the amino acid sequence of SEQ ID NO:9.

In some embodiments, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, relative to SEQ ID NO: 41 or 42, Chimeric receptors are provided comprising an amino acid sequence having at least approximately any one of 97%, 98%, 99%, or 100% sequence identity. In some embodiments, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, relative to SEQ ID NO: 43 or 44, An isolated nucleic acid is provided comprising a nucleic acid sequence having at least about any one of 97%, 98%, 99%, or 100% sequence identity.

The IL-3 (interleukin-3) gene is mapped onto chromosome 5, which encodes a 152 amino acid long protein. IL-3 is a cytokine that can support a wide range of cellular activities such as cell activity, such as cell growth, differentiation and apoptosis. IL-3 acts by binding to the interleukin-3 receptor (IL-3R), also known as the CD123 antigen. IL-3R is a heterodimer comprising a ligand-specific alpha subunit and a signal signaling beta subunit, which subunits are receptors for IL-3, colony stimulating factor 2 (CSF2/GM-CSF), and interleukin 5 It is shared by receptors for (IL5). The activation of IL-3R results in phosphorylation of the βc chain, recruitment of SH2-containing adapter molecules such as Vav1, and downstream signaling through the Jak2/STAT5 and Ras/MAPK pathways.

IL-3R is a 75 kD glycoprotein and becomes 43 kD when hydrolyzed by N-glycosidase. IL-3R has three extracellular domains responsible for specific binding to IL-3, a transmembrane domain, and a short intercellular domain that is indispensable for intracellular signaling (Sato et al. 1993). IL-3R is a heterodimeric receptor with low affinity and high specificity for IL-3. When binding to IL-3, IL-3R is activated, and cell proliferation and survival are promoted (Liu et al. 2015).

CD123 is overexpressed in AML blasts ( eg , bone marrow). In 75-89% of AML patients, AML progenitors and leukemia stem cells (LSCs) express CD123. In sharp contrast, the expression of CD123 in normal hematopoietic stem cells (HSCs) is low or undetectable (Frankel et al. 2014; Jordan et al. 2000). In addition to AML, CD123 is also overexpressed in a variety of hematologic malignancies, including B cell lineage acute lymphoblastic leukemia, chronic myelogenous leukemia, plasma cell dendritic cell neoplasms and hairy cell leukemia (Munoz et al. 2001). This expression profile makes CD123 a useful biomarker in the clinical diagnosis, prognosis and intervention of the disease. Currently, early-stage clinical trials have demonstrated that CD123-targeted therapy is safe and has no significant side effects on hematopoiesis. The anti-leukemia activity of CD123-targeted therapy in humans is still being studied.

Dimerization motif

In some embodiments, the extracellular domain comprises a dimerization motif and a single NKG2D domain. In some embodiments, the extracellular domain comprises a second antigen binding domain, a single NKG2D domain, and a dimerization motif disposed therebetween. The dimerization motif promotes dimerization of the two polypeptide chains in the chimeric receptor, thereby facilitating the formation of the NKG2D homodimer and the binding of the NKG2D homodimer to the NKG2D ligand. Suitable dimerization motifs are known in the art. In some embodiments, the dimerization motif is a leucine zipper. In some embodiments, the leucine zipper comprises the amino acid of SEQ ID NO: 10. In some embodiments, the dimerization motif is a cysteine zipper. Exemplary cysteine zippers are known in the art. For example, Guilaume et al. See (2015) PLoS ONE 10(6): e0128779.

Peptide linker

In some embodiments, the NKG2D domain and the second antigen binding domain ( eg , IL-3 domain) are fused to each other via a peptide linker. In some embodiments, the first NKG2D domain and the second NKG2D domain are fused to each other via a peptide linker. The different domains of the chimeric receptor can also be fused to each other via a peptide linker. The peptide linkers connecting different domains can be the same or different.

Each peptide linker in the chimeric receptor can have the same or different lengths depending on the structural and/or functional characteristics of the various domains. Each peptide linker can be independently selected and optimized. The length, softness, and/or other properties of the peptide linker(s) used in the chimeric receptor include, but are not limited to, affinity, specificity, or antigen-antibody binding ability for one or more specific antigens or epitopes. The degree can be affected. For example, a longer peptide linker may be chosen so that the two adjacent domains do not sterically interfere with each other. In some embodiments, a short peptide linker may be placed between the transmembrane domain of the chimeric receptor and the intracellular signaling domain. In some embodiments, the peptide linker comprises flexible moieties (eg, glycine and serine) such that adjacent domains are free to move relative to each other. For example, a glycine-serine doublet may be a suitable peptide linker.

The peptide linker can have any suitable length. In some embodiments, the peptide linker is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 25, 30, 35, 40, 50, 75, 100 or more amino acids in length. In some embodiments, the peptide linker is at most about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, It is 8, 7, 6, 5 or less amino acids long. In some embodiments, the length of the peptide linker is about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids. Amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 Can be any of dog amino acids, or about 1 amino acid to about 100 amino acids.

The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived exclusively from the hinge region of a heavy chain antibody can be used as a linker. See, for example, WO1996/34103. In some embodiments, the peptide linker is a soft linker. Exemplary flexible linkers include glycine polymer (G) _n (SEQ ID NO: 28), glycine-serine polymer (e.g., (GS) _n (SEQ ID NO: 29), (GSGGS) _n (SEQ ID NO: 30), ( GGGS) _n (SEQ ID NO: 31), and (GGGGS) _n (SEQ ID NO: 32), wherein n is an integer of at least 1), glycine-alanine polymer, alanine-serine polymer, and other softness known in the art Includes a linker. In some embodiments, the peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 12-15.

Transmembrane domain

The chimeric receptors, including the multispecific chimeric receptor of the present application, the first chimeric receptor and the second chimeric receptor of the dual chimeric receptor system, have a transmembrane domain that can be directly or indirectly fused to the extracellular antigen-binding domain. Include. The transmembrane domain can be derived from natural or synthetic sources. As used herein, “transmembrane domain” refers to any protein structure that is thermodynamically stable in cell membranes, preferably eukaryotic cell membranes. Transmembrane domains suitable for use in the chimeric receptors described herein can be obtained from native proteins. Alternatively, it may be a synthetic non-natural protein segment, for example a hydrophobic protein segment that is thermodynamically stable in the cell membrane.

Transmembrane domains are classified based on the three-dimensional structure of the transmembrane domain. For example, transmembrane domains can form an alpha helix, a complex of one or more alpha helices, a beta-barrel, or any other stable structure that can span the phospholipid bilayer of a cell. In addition, the transmembrane domains can be classified based on or alternatively based on the topology of the transmembrane domain including the number of times the transmembrane domain passes across the membrane and the orientation of the protein. For example, a single-pass membrane protein traverses the cell membrane once, and a multi-pass membrane protein traverses the cell membrane at least two times ( eg , 2, 3, 4, 5, 6, 7 or more times). Membrane proteins can be defined as type I, type II or type III depending on the topology of the terminal and transmembrane segment(s) to the inside and outside of the cell. Type I membrane proteins have a single membrane-spanning region, the N-terminus of the protein being oriented so that it is on the extracellular side of the lipid bilayer of the cell, and the C-terminus of the protein is on the cytoplasmic side. Type II membrane proteins have a single membrane-spanning region, but the C-terminus of the protein is oriented so that it is on the extracellular side of the lipid bilayer of the cell, and the N-terminus of the protein is on the cytoplasmic side. . Type III membrane proteins have a number of membrane-spanning segments and can be further classified based on the number of transmembrane segments and the location of the N- and C-terminals.

In some embodiments, the transmembrane domain of the chimeric receptor described herein is derived from a type I single-pass membrane protein. In some embodiments, the transmembrane domains of a multi-pass membrane protein are compatible for use with the chimeric receptor described herein. Multi-pass membrane proteins may comprise complex (at least 2, 3, 4, 5, 6, 7 or more) alpha helical or beta sheet structures. Preferably, the N-terminus and C-terminus of the multi-pass membrane protein are on opposite sides of the lipid bilayer, e.g. , the N-terminus of the protein is on the cytoplasmic side of the lipid bilayer, and the C-terminus of the protein Exists on the extracellular side.

In certain embodiments, the transmembrane domain of the chimeric receptor is a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160 , CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR , PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or a transmembrane domain selected from the transmembrane domain of the alpha, beta or zeta chain of NKG2C. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152 and PD1.

In some embodiments, the transmembrane domain is derived from CD28. In some embodiments, the transmembrane domain is derived from CD8α. In some embodiments, the transmembrane domain is derived from the full length transmembrane domain of CD8α. In some embodiments, the transmembrane domain is derived from a CD8α truncated transmembrane domain. In some embodiments, the transmembrane domain is a transmembrane domain of CD8α, comprising the amino acid sequence of SEQ ID NO: 4 or 45.

The transmembrane domain for use in the chimeric receptor described herein may also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least approximately 20 amino acids, such as at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more. It is an amino acid. Examples of synthetic transmembrane domains are in the art, eg, US Patent No. 7,052,906 B1 and PCT Publication No. It is known from WO 2000/032776 A2, the content of which is incorporated herein by reference.

The transmembrane domain may include a transmembrane region and a cytoplasmic region located at the C-terminus of the transmembrane domain. The cytoplasmic region of the transmembrane domain comprises three or more amino acids, and in some embodiments, it supports orientation of the transmembrane domain in the lipid bilayer. In some embodiments, one or more cysteine residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of the chimeric receptor comprises an artificial hydrophobic sequence. For example, the triplet of phenylalanine, tryptophan and valine can be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region mostly comprises hydrophobic amino acid residues such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence. The hydropathy or hydrophobic or hydrophilic properties of a protein or protein segment can be assessed by any method known in the art, for example Kyte and Doolittle numerical analysis.

Intracellular signaling domain

The chimeric receptors including the multispecific chimeric receptor of the present application, the first chimeric receptor and the second chimeric receptor of the dual chimeric receptor system include an intracellular signaling domain. The intracellular signaling domain is responsible for the activation of at least one of the normal effector functions of immune effector cells expressing the chimeric receptor. The term “effector function” refers to the specialized function of a cell. The effector function of T cells can be, for example, cytolytic activity or helper activity, including the secretion of cytokines. Thus, the term “cytoplasmic signaling domain” refers to a portion of a protein that transforms the effector function signal in question and directs the cell to perform a specific function. Usually the entire cytoplasmic signaling domain can be used, but in many cases it is not necessary to use the entire chain. As long as the truncated portion of the cytoplasmic signaling domain is used, it can be used in place of the intact chain if such truncated portion carries intact effector function signals. Thus, the term cytoplasmic signaling domain is meant to include any truncated portion of the cytoplasmic signaling domain sufficient to transform the effector function signal in question.

In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the chimeric receptor comprises a signaling domain consisting essentially of the primary intracellular signaling domain of an immune effector cell. “Primary intracellular signaling domain” refers to a cytoplasmic signaling sequence that acts in a stimulus mode that induces immune effector function. In some embodiments, the primary intracellular signaling domain contains an immunoreceptor tyrosine-based activation motif, or a signaling motif known as ITAM. “ITAM”, as used herein, is a conserved protein motif typically present in the tail of a signaling molecule expressed in many immune cells, the motif being separated by 6-8 amino acids, the amino acid sequence YxxL It may contain two repeats of /I, where each x is independently any amino acid, making the conserved motif YxxL/Ix(6-8)YxxL/I. ITAMs in the signaling molecule signal within the cell Important for transformation, which is mediated at least in part by phosphorylation of tyrosine residues in ITAM followed by activation of the signaling molecule ITAMs can also function as docking sites for other proteins involved in the signaling pathway. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3ζ, FcR gamma (FCER1G), FcR beta (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Included.

In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain consists of the cytoplasmic signaling domain of CD3ζ. In some embodiments, the primary intracellular signaling domain is the cytoplasmic signaling domain of wild-type CD3ζ. In some embodiments, the primary intracellular signaling domain of wild-type CD3ζ comprises the amino acid sequence of SEQ ID NO: 6.

Co-stimulatory signaling domain

In addition to stimulation of antigen-specific signals, many immune effector cells require co-stimulation to promote cell proliferation, differentiation and survival, as well as to activate effector functions of cells. In some embodiments, the intracellular signaling domain comprises at least one co-stimulatory signaling domain. The term “co-stimulatory signaling domain” as used herein refers to at least a portion of a protein that mediates signal transduction in a cell, such as to elicit an immune response, such as effector function. The co-stimulatory signaling domain of the chimeric receptor may be a cytoplasmic signaling domain of a co-stimulatory protein, which converts the signal and converts the signal, and immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. ) Regulates the response mediated by the "co-stimulatory signaling domain" may be the cytoplasmic part of the co-stimulatory molecule The term "co-stimulatory molecule" specifically binds to a co-stimulatory ligand and is thereby Because of the co-stimulatory response by the immune cells, such as proliferation and survival, it refers to a cognate binding partner on immune cells (such as T cells) that mediate reactions including, but not limited to.

In some embodiments, the intracellular signaling domain comprises a single co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (such as any of about 2, 3, 4, or more) co-stimulatory signaling domains. In certain embodiments, the intracellular signaling domain comprises two or more identical co-stimulatory signaling domains, eg, two copies of the co-stimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains of different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein. do. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3ζ) and one or more co-stimulatory signaling domains. In some embodiments, the one or more co-stimulatory signaling domains and the primary intracellular signaling domain (such as the CD3ζ cytoplasmic signaling domain) are fused to each other through an optional peptide linker. The primary intracellular signaling domain, and one or more co-stimulatory signaling domains can be aligned in any suitable order. In some embodiments, the one or more co-stimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as the CD3ζ cytoplasmic signaling domain). Multiple co-stimulatory signaling domains can provide an additive or synergistic stimulating effect.

Activation of a co-stimulatory signaling domain in a host cell ( e.g. , an immune cell) can induce an increase or decrease in the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and/or cytotoxicity from that cell. . The co-stimulatory signaling domain of any co-stimulatory molecule may be compatible for use in the chimeric receptors described herein. The type(s) of the co-stimulatory signaling domain are factors, such as the type of immune effector cell in which the effector molecule is expressed ( e.g. , T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune function. Can be selected based on body function ( eg ADCC effect). Exemplary co-stimulatory signaling domains for use in chimeric receptors may be the cytoplasmic signaling domain of a co-stimulatory protein, including but not limited to: Components of the B7/CD28 family ( e.g. , B7 -1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24 /VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, and PDCD6); Components of the TNF superfamily ( e.g. , 4-1BB/TNFSF9/CD137, 4-1BB ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14, Lymphotoxin-alpha/TOX40 , OX40 ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF13B, TL1A/TNFSF15, TNF-alpha, and TNF RII/TNFRSF1B); Components of the SLAM family ( e.g. , 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB -A/SLAMF6, and SLAM/CD150); And any other co-stimulatory molecules, such as CD2, CD7, CD53, CD82/Kai-1, CD90/Thy1, CD96, CD160, CD200, CD300a/LMIR1, HLA class I, HLA-DR, Ikaros, integrin alpha 4 /CD49d, integrin alpha 4 beta 1, integrin alpha 4 beta 7/LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1/CLEC7A, DPPIV/CD26, EphB6, TIM-1/KIM-1 /HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen-1 (LFA-1), and NKG2C.

In some embodiments, the one or more co-stimulatory signaling domains are CD27, CD28, 4-1BB ( e.g. , CD137), ICOS, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA -1), is selected from the group consisting of a ligand that specifically binds to CD2, CD7, LIGHT, NKG2C, B7-H3 and CD83.

In some embodiments, the intracellular signaling domain in the chimeric receptor of the present application comprises a co-stimulatory signaling domain derived from CD28. In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3ζ and a co-stimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain in the chimeric receptor of the present application comprises a co-stimulatory signaling domain derived from 4-1BB ( eg , CD137). In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3ζ and a co-stimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain of 4-1BB comprising the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the intracellular signaling domain in the chimeric receptor of the present application comprises a co-stimulatory signaling domain of CD28 and a co-stimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3ζ, a co-stimulatory signaling domain of CD28, and a co-stimulatory signaling domain of 4-1BB. In certain embodiments, the intracellular signaling domain comprises a polypeptide in the N-terminal to C-terminal direction, comprising: a co-stimulatory signaling domain of CD28, a co-stimulatory signaling of 4-1BB. Domain, and the cytoplasmic signaling domain of CD3ζ.

Variants of any of the co-stimulatory signaling domains described herein are within the scope of the present invention provided that the co-stimulatory signaling domain is capable of modulating the immune response of immune cells. In some embodiments, the co-stimulatory signaling domains contain up to 10 amino acid residue variations ( eg , 1, 2, 3, 4, 5, or 8) when compared to the wild-type counterpart. Co-stimulatory signaling domains comprising one or more amino acid variants may also be referred to as variants. Due to mutations in the amino acid residues of the co-stimulatory signaling domain, it can lead to an increase in signaling transduction and enhanced stimulation of the immune response when compared to a co-stimulatory signaling domain that does not contain the mutation. Mutations in the amino acid residues of the co-stimulatory signaling domain can result in a reduction in signaling transduction and reduced stimulation of the immune response when compared to a co-stimulatory signaling domain that does not contain the mutation.

Hinge area

The chimeric receptors, including the multispecific chimeric receptor of the present application, the first chimeric receptor and the second chimeric receptor of the dual chimeric receptor system, may include a hinge region located between the extracellular antigen-binding domain and the transmembrane domain. . The hinge region is an amino acid segment generally found between the two domains of a protein, and can allow the flexibility of the protein and the movement of one or both domains with respect to each other. For the transmembrane domain of this effector molecule, any amino acid sequence that provides this flexibility and movement of the extracellular antigen binding domain can be used.

The hinge region may contain about 10-100 amino acids, such as about 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. In certain embodiments, the amino acid length of the hinge region is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids.

In some embodiments, the hinge region is a hinge region of a naturally occurring protein. The hinge region of any protein known in the art to include the hinge region is suitable for use in the chimeric receptor described herein. In some embodiments, the hinge region is at least a portion of the hinge region of a naturally occurring protein, which confers flexibility to the chimeric receptor. In some embodiments, the hinge region is derived from CD8α. In some embodiments, the hinge region is a portion of the hinge region of CD8α, such as a fragment containing at least 15 ( eg , 20, 25, 30, 35, or 40) contiguous amino acids of the hinge region of CD8α. In some embodiments, the hinge region of CD8α comprises the amino acid sequence of SEQ ID NO: 3.

The hinge regions of antibodies, such as IgG, IgA, IgM, IgE, or IgD antibodies, are suitable for use in the chimeric receptors described herein. In some embodiments, the hinge region is a hinge region connecting the constant domains CH1 and CH2 of the antibody. In some embodiments, the hinge region of the antibody comprises a hinge region of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge region comprises a hinge region of the antibody and a CH3 constant region of the antibody. In some embodiments, the hinge region comprises a hinge region of the antibody and CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises a hinge region of the antibody and CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises a hinge region of the antibody and a CH3 constant region of an IgG1 antibody.

Non-naturally occurring peptides can also be used as hinge regions for the chimeric receptors described herein. In some embodiments, the hinge region between the C-terminus and the transmembrane domain of the extracellular ligand-binding domain of the Fc receptor is a peptide linker, such as a (GxS)n linker, wherein x and n are independently 3 and 12 It may be an integer between, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

Signal peptide

The chimeric receptors, including the multispecific chimeric receptor of the present application, the first chimeric receptor and the second chimeric receptor of the dual chimeric receptor system, are signal peptides (also known as signal sequences) at the N-terminus of the polypeptide(s). ) Can be included. Typically, a signal peptide is a peptide sequence that targets a polypeptide to a desired site in a cell. In some embodiments, the signal peptide will target the effector molecule to the secretory pathway of the cell and allow for integration and immobilization of the effector molecule into the lipid bilayer. Signal peptides comprising signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences suitable for use in the chimeric receptors described herein will be apparent to those of skill in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, IL-3, and IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8α. In some embodiments, the signal peptide of IL-3 comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the signal peptide of CD8α comprises the amino acid sequence of SEQ ID NO: 2.

III. Engineered immune effector cells

Host cells (such as engineered immune effector cells) comprising any one of the chimeric receptors, multispecific chimeric receptors or dual chimeric receptor systems described herein are further provided herein.

Thus, in some embodiments, an engineered immune effector cell (such as a T cell) comprising a chimeric receptor is provided, wherein the chimeric receptor comprises: (a) an extracellular domain comprising an NKG2D domain; (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the extracellular domain comprises a first NKG2D domain and a second NKG2D domain.

In some embodiments, an engineered immune effector cell (such as a T cell) comprising a multispecific ( eg , bispecific) chimeric receptor is provided, wherein the chimeric receptor comprises: (a) NKG2D An extracellular domain comprising a domain and a second antigen binding domain ( eg , IL-3 domain); (b) transmembrane domain; And (c) an intracellular signaling domain.

In some embodiments, an engineered immune effector cell (such as a T cell) comprising a chimeric receptor comprising a polypeptide chain is provided, wherein the polypeptide chain comprises: (a) a first NKG2D domain, a second 2 NKG2D domain, and an extracellular domain comprising a CD123 binding domain (eg, IL-3 domain); (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the CD123 binding domain is an anti-CD123 antibody fragment ( eg , scFv or VHH). In some embodiments, the CD123 binding domain is an IL-3 domain. In some embodiments, the polypeptide chain in the N-terminal to C-terminal direction, comprises: an IL-3 domain, a first peptide linker, a first NKG2D domain, a second peptide linker, a second NKG2D domain, CD8α hinge region, CD8α transmembrane domain, co-stimulatory signaling domain derived from 4-1BB, and primary intracellular signaling domain derived from CD3ζ.

In some embodiments, an engineered immune effector cell (such as a T cell) comprising a multispecific ( e.g. , bispecific) chimeric receptor comprising a first polypeptide chain and a second polypeptide chain is provided, wherein Each of the chains comprises: (a) an extracellular domain including an NKG2D domain and a CD123 binding domain ( eg , an IL-3 domain); (b) transmembrane domain; And (c) an intracellular signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is crosslinked to the NKG2D domain of the second polypeptide chain through one or more disulfide bonds. In some embodiments, each of the first polypeptide chain and the second polypeptide chain in the N-terminal to C-terminal direction comprises: an IL-3 domain, a peptide linker, an NKG2D domain, a CD8α hinge region, a CD8α transmembrane. Domain, a co-stimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the extracellular domain further comprises a dimerization motif ( eg , a leucine zipper or a cysteine zipper) disposed between the NKG2D domain and the CD123 binding domain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain in an N-terminal to C-terminal direction comprises: an IL-3 domain, a leucine zipper, a NKG2D domain, a CD8α hinge region, a CD8α transmembrane. Domain, a co-stimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ.

In some embodiments, an engineered immune effector cell (such as a T cell) is provided comprising a dual chimeric receptor system comprising: (i) a first comprising a first polypeptide chain and a second polypeptide chain. Each chimeric receptor chain contains: (a) an extracellular domain comprising a first NKG2D domain; (b) a first transmembrane domain; And (c) a first intracellular signaling domain; And (ii) a second chimeric receptor comprising a third polypeptide chain comprising: (a) a second extracellular domain comprising a second antigen binding domain; (b) a second transmembrane domain; And optionally (c) a second intracellular signaling domain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain in the N-terminal to C-terminal direction comprises: NKG2D domain, CD8α hinge region, CD8α transmembrane domain, derived from 4-1BB. A co-stimulatory signaling domain, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the second chimeric receptor comprises a polypeptide comprising, in the N-terminal to C-terminal direction, an IL-3 domain, a CD8α hinge region, a CD8α transmembrane domain, and optionally 4- Co-stimulatory signaling domain derived from 1BB.

In some embodiments, an engineered immune effector cell (such as a T cell) is provided comprising a dual chimeric receptor system comprising: (i) a first chimeric receptor comprising a first polypeptide chain, wherein the chain is It includes: (a) a first extracellular domain comprising a first NKG2D domain and a second NKG2D domain; (b) a first transmembrane domain; And (c) a first intracellular signaling domain; And (ii) a second chimeric receptor comprising a second polypeptide chain, wherein the chain comprises: (a) a second extracellular domain comprising a second antigen binding domain; (b) a second transmembrane domain; And optionally (c) a second intracellular signaling domain. In some embodiments, a first chimeric receptor N-terminal to C-terminal direction, comprises a polypeptide comprising: a first NKG2D domain, a second NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain, 4- The co-stimulatory signaling domain derived from 1BB, and the primary intracellular signaling domain derived from CD3ζ. In some embodiments, the second chimeric receptor comprises a polypeptide comprising, in the N-terminal to C-terminal direction, an IL-3 domain, a CD8α hinge region, a CD8α transmembrane domain, and optionally 4- Co-stimulatory signaling domain derived from 1BB.

In some embodiments, the engineered immune effector cells express one or more immunomodulatory agents. In some embodiments, the engineered mammalian cell comprises a heterologous nucleic acid encoding the immunomodulatory agent. In some embodiments, the heterologous nucleic acid encoding the immunomodulatory agent is on a vector encoding a chimeric receptor, multispecific chimeric receptor or dual chimeric receptor system described herein. In some embodiments, the immunomodulatory agent is a modulator of Runx3. In some embodiments, the modulator is a polypeptide, such as a polypeptide derived from Runx3 or MITF. In some embodiments, the modulator is RNA, such as miRNA or siRNA. Runx3 can aid in T cell homing and invasion into solid tumors, which is an important factor in successful T-cell mediated immunotherapy.

vector

This application provides vectors for cloning and expressing any one of the chimeric receptor, multispecific chimeric receptor or dual chimeric receptor systems described herein. In some embodiments, the vector is suitable for replication and integration in eukaryotic cells such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. Viral vector technology is well known in the art and, for example, Sambrook et al . (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals.

A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The heterologous nucleic acid may be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the engineered mammalian cells in vitro or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying chimeric receptors, multispecific chimeric receptors or dual chimeric receptor systems can be packaged according to protocols known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells (such as primary human T cells) using methods known in the art. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of the transgene and its propagation in its progeny cells. to be. Lentiviral vectors are also low immunogenic and are capable of transducing non-proliferating cells.

In some embodiments, the vector comprises any one of a nucleic acid encoding a chimeric receptor, a multispecific chimeric receptor, or a dual chimeric receptor system described herein. Nucleic acids can be cloned into vectors using any known molecular cloning method in the art, including, for example, using restriction endonuclease sites and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. Various promoters have been studied for gene expression in mammalian cells, and any promoter known in the art can be used in the present invention. Promoters can be broadly classified as constitutive or regulated promoters, such as inducible promoters.

In some embodiments, the nucleic acid encoding the chimeric receptor, multispecific chimeric receptor or dual chimeric receptor system is operably linked to a constitutive promoter. Constituent promoters allow heterologous genes (also called transgenes) to be expressed constitutively in the host cell. Exemplary constitutive promoters contemplated herein include cytomegalovirus (CMV) promoter, human elongation factor-1 alpha (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), monkey virus 40 early promoter (SV40), and the chicken β-actin promoter bound to CMV early enhancer (CAGG), but is not limited thereto. The efficiency of such constitutive promoters in inducing transgene expression has been widely compared in numerous studies. For example, Michael C. Milone et al . compared the efficiency of CMV, hEF1α, UbiC and PGK to induce chimeric receptor expression in primary human T cells, and the hEF1α promoter not only induces transgene expression to the highest level, but also CD4 and It was concluded that it was optimally maintained in CD8 human T cells (Molecular Therapy, 17(8): 1453-1464 (2009)).

In some embodiments, the nucleic acid encoding the chimeric receptor, multispecific chimeric receptor or dual chimeric receptor system is operably linked to an inducible promoter. Inducible promoters belong to the category of regulated promoters. The inducible promoter is one or more conditions, such as the physical condition of the engineered immune effector cell, the microenvironment, or the physiological state of the engineered immune effector cell, an inducer ( such as an inducer), or its It can be induced by combination. In some embodiments, the inducing condition does not induce expression of an endogenous gene in the engineered mammalian cell, and/or in a subject receiving the pharmaceutical composition. In some embodiments, the induction condition is selected from the group consisting of an inducing agent, irradiation (such as ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and activation state of engineered mammalian cells. .

In some embodiments, the vector also contains a selectable marker gene or reporter gene to select cells expressing the chimeric receptor, multispecific chimeric receptor or dual chimeric receptor system in a population of host cells transfected via a lentiviral vector. Contains. Both the selectable marker and reporter gene can be flanked by appropriate regulatory sequences to allow expression in the host cell. For example, the vector may contain a transcription and translation terminator, an initiation sequence, and a promoter useful for regulating the expression of nucleic acid sequences.

Immune effector cells

“Immune effector cells” are immune cells capable of performing immune effector functions. In some embodiments, the immune effector cell expresses at least FcγRIII and performs ADCC effector function. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, single cells, cytotoxic T cells, neutrophils, and eosinophils.

In some embodiments, the immune effector cell is a T cell. In some embodiments, the T cell is CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-, or a combination thereof. In certain embodiments, the T cell is IL-2, TFN, and/or when the chimeric receptor, multispecific chimeric receptor, or dual chimeric receptor system is expressed and bound to a target cell, such as a CD20+ or CD19+ tumor cell. Or produce TNF. In some embodiments, CD8+ T cells lyse antigen-specific target cells when the chimeric receptor, multispecific chimeric receptor or dual chimeric receptor system is expressed and bound to the target cell.

In some embodiments, the immune effector cell is an NK cell. In other embodiments, the immune effector cells may be an established cell line, for example NK-92 cells.

In some embodiments, the immune effector cells are differentiated stem cells, such as hematopoietic stem cells, pluripotent stem cells, iPS, or embryonic stem cells.

The engineered immune effector cells are prepared by introducing the chimeric receptor, multispecific chimeric receptor or dual chimeric receptor system into the immune effector cells, such as T cells. In some embodiments, the chimeric receptor, multispecific chimeric receptor or dual chimeric receptor system can be introduced into an immune effector cell by transfecting an isolated nucleic acid described herein or a vector described herein. In some embodiments, the chimeric receptor was inserted into the multi-specific chimeric receptor, or dual chimeric receptor system cell protein membrane, the cells are introduced into the by-pass a microfluidic system, For instance CELL SQUEEZE ^®, immune effector cells (See, for example, US Patent Application Publication No. 20140287509).

Methods of introducing vectors or isolated nucleic acids into mammalian cells are known in the art. The described vectors can be delivered into immunoeffector cells by physical, chemical or biological methods.

Physical methods of introducing the vector into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. For example, Sambrook et al . (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, the vector is introduced into the cell by electroporation.

Biological methods of introducing such vectors into immune effector cells include the use of DNA vectors and RNA vectors. Viral vectors have become the most widely used method of inserting genes into mammalian cells such as human cells.

Chemical means for introducing the vector into immune effector cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based, including oil-in-water emulsions, micelles, mixed micelles and liposomes. Including the system. An exemplary colloidal system for use as an in vitro delivery vehicle is liposomes (eg, artificial membrane vesicles).

In some embodiments, RNA molecules encoding chimeric receptors, multispecific chimeric receptors or dual chimeric receptor systems described herein are prepared by conventional methods ( e.g. , in vitro transcription), and known methods, such as mRNA is introduced into immune effector cells through electroporation. For example , Rabinovich et al. , Human Gene Therapy 17:1027-1035.

In some embodiments, the transduced or transfected immune effector cell is propagated ex vivo following introduction of the vector or the isolated nucleic acid. In certain embodiments, the transduced or transfected immune effector cells are at least about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. It is cultured to propagate during the period. In some embodiments, the transduced or transfected immune effector cells can be further evaluated or screened to select the engineered mammalian cells.

Reporter genes can be used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present or expressed in a recipient organism or tissue, and is a gene encoding a property that is readily detectable by expression of the polypeptide, such as a polypeptide exhibited by enzymatic activity. The expression of the reporter gene is analyzed at an appropriate time after the DNA is introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase or green fluorescent protein gene ( eg , Ui-Tei et al . FEBS Letters 479: 79-82 (2000)). Suitable expression systems are known and can be prepared or commercially available using known techniques.

Other methods of identifying the presence of nucleic acids encoding the chimeric receptor, multispecific chimeric receptor or dual chimeric receptor system in the engineered immune effector cell include, for example, molecular biological assays known in the art, such as Southern and Northern blotting, RT-PCR and PCR; Biochemical assays, such as detection of the presence or absence of a particular peptide, such as by immunological methods (such as ELISAs and Western blots) are included.

Source of T cells

Prior to expansion and genetic modification of T cells, a source of T cells is obtained from the individual. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue and tumors. In some embodiments, multiple T cell lines available in the art can be used. In some embodiments, T cells can be obtained from blood units collected from a subject using any number of techniques known to those of skill in the art, such as FICOLL™ isolation. In some embodiments, cells from the subject's circulating blood are obtained by apheresis. Apheresis products typically contain lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets. In some embodiments, cells collected by apheresis are washed to remove plasma fractions, and the cells can be placed in a buffer or medium suitable for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash liquor may be lacking in calcium, lacking in magnesium, or may lack many, if not these two divalent cations. Again, surprisingly, the initial activation step in the absence of calcium leads to magnified activation. Methods known in the art using a semi-automated “flow-through” centrifuge (eg, Cobe 2991 Cell Processor, Baxter CytoMate or Haemonetics Cell Saver 5) where the washing step is performed according to the manufacturer's instructions. Those skilled in the art will readily appreciate that it can be made according to. After washing, the cells can be resuspended in various biocompatible buffers, such as PBS without Ca ²⁺ -free, Mg ²⁺ -free, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components can be removed from the apheresis sample and the cells can be resuspended directly in the culture medium.

In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example by centrifugation through a PERCOLL™ gradient or countercurrent centrifugal elution. Certain subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in some embodiments, anti-CD3/anti-CD28 (e.g., 3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time sufficient for positive selection of the desired T cells. By incubating with, T cells are isolated. In some embodiments, the period is about 30 minutes. In a further embodiment, the period ranges from 30 minutes to 36 hours or longer and all integer values in between. In further embodiments, the period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the period is 10 to 24 hours. In some embodiments, the incubation period is 24 hours. Using longer incubation times, such as 24 hours, to isolate T cells from leukemia patients can increase cell yield. Compared to other cell types, to isolate T cells in any situation where there are few T cells, for example, isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or from immune-compromised individuals is more Long incubation periods can be used. In addition, the use of longer incubation times can increase the capture efficiency of CD8+ T cells. Thus, by simply shortening or prolonging the time for which T cells bind to CD3/CD28 beads, and/or by increasing or decreasing the ratio of T cells to beads (as further described herein), Subpopulations may be selected preferentially, or against, at or at different times during the initiation of culture or during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surfaces, a subpopulation of T cells is preferentially selected at the initiation of culture or other desirable time point, or aagainst Can be chosen. One of skill in the art will recognize that multiple rounds of selection may also be used. In some embodiments, it may be desirable to perform a selection procedure and use “unselected” cells in the activation and expansion process. Cells that are “unselected” may also undergo further rounds of selection as well.

The enrichment of the T cell population by negative selection can be achieved with a combination of antibodies directed against a specific surface marker for negatively selected cells. One method is cell sorting and/or selection through negative magnetic immunosorbent or flow cytometry, using a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, monoclonal antibody cocktails typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8. In certain embodiments, it may be desirable to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+ and FoxP3+. Alternatively, in certain embodiments, T regulatory cells can be depleted by anti-C25 conjugated beads or other similar selection method.

In order to isolate the desired cell population by positive or negative selection, the concentration of cells and surfaces (eg, particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (ie, increase the concentration of cells) to ensure maximum contact of the cells and beads. For example, in one embodiment, a concentration of 2 billion cells/mL is used. In one embodiment, a concentration of 100 million cells/mL is used. In a further embodiment, more than 100 million cells/mL are used. In a further embodiment, a cell concentration of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells/mL is used. In still another embodiment, a cell concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells/mL is used. In additional embodiments, concentrations of 125 million or 150 million cells/mL may be used. The use of high concentrations can increase cell yield, cell activation and cell expansion. In addition, the use of high cell concentrations can weakly express the target antigen of interest, such as CD28-negative T cells, or cells that can be weakly expressed from samples in which many tumor cells are present (i.e. leukemia blood, tumor tissue, etc.). Makes it possible to capture more efficiently. These cell populations may have therapeutic value and would be desirable to obtain. For example, the use of high concentrations of cells generally allows more efficient selection of CD8+ T cells with weaker CD28 expression.

In some embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surfaces (eg, particles such as beads), interactions between particles and cells are minimized. This selects cells that express a large amount of the desired antigen to be bound to the particle. For example, CD4+ T cells express CD28 at higher levels and are captured more efficiently than CD8+ T cells at dilution concentrations. In some embodiments, the concentration of cells used is 5x10 ⁶ /mL. In some embodiments, the concentration used can be between about 1×10 ⁵ /mL and 1×10 ⁶ /mL and any integer value therebetween.

In some embodiments, cells may be cultured in a rotator at 2-10° C. or room temperature at various rates and for various lengths of time.

T cells for stimulation can also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogeneous product by removing granulocytes and some degree of monocytes from the cell population. After a washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and may be useful in this context, one method is PBD containing 20% DMSO and 8% human serum albumin, or 10% dextran 40 and 5%. Dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5 % DMSO, or a suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, then the cells are frozen at -80° C. at a rate of 1° per minute, and stored in liquid nitrogen. It is stored in the vapor phase of the tank. Other controlled freezing methods can be used, as well as immediate or uncontrolled freezing at -20°C in liquid nitrogen.

In some embodiments, cryopreserved cells were thawed, washed, and rested for 1 hour at room temperature prior to activation as described herein.

Collection of a blood sample or apheresis product from a subject prior to a time when expanded cells as described herein may be needed is also contemplated in this application. As such, the source of cells to be expanded can be collected at any time point needed, and the desired cells, such as T cells, can be isolated and frozen to benefit from any number of diseases or diseases that can benefit from T cells as described herein. It can be used later in T cell therapy for the condition. In one embodiment, the blood sample or component collection is generally taken from a healthy subject. In certain embodiments, the blood sample or component collection is taken from a generally healthy subject at risk of developing the disease, but not yet developing the disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, T cells are expanded, frozen, and can be used later. In certain embodiments, a sample is collected from the patient immediately after diagnosis of a particular disease as described herein, but prior to any treatment. In a further embodiment, the cells are isolated from a blood sample or component collection from a subject prior to any number of relevant treatment modalities below: natalizumab, epalizumab, antiviral, chemotherapy, radiation, immunosuppressant such as cyclosporine, Agents such as azathioprine, methotrexate, mycophenolate and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytosan, fludarabine, cyclosporine, FK506, rapamycin, myco Treatment and irradiation with agents such as phenolic acid, steroids, FR901228. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506), or p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Liu et al., Cell 66:807-815, 1991; Henderson Et al., Immun 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In a further embodiment, the cells are isolated from the patient, and bone marrow or stem cell transplantation, fludarabine, external-beam radiation therapy (XRT), a chemotherapeutic agent such as cyclophosphamide, or a chemotherapeutic agent such as OKT3 or CAMPATH. Frozen for later use (eg, before or at the same time or after) with a T cell ablative therapy regimen using the antibody. In another embodiment, the cells can be frozen for later use in treatment following a B-cell ablation therapy such as an agent that reacts with CD20, for example Rituxan.

In some embodiments, T cells are obtained directly from the patient after treatment. In this regard, the quality of the T cells obtained may be optimal, or their ability to expand ex vivo, after treatment with certain cancer treatments, in particular drugs that damage the immune system, during the period when the patient normally recovers from treatment immediately after treatment. This can be improved. Likewise, after ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, during this recovery phase, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells or other cells of the hematopoietic lineage. Moreover, in certain embodiments, the mobilization (e.g., mobilization by GM-CSF) and conditioning therapy may be used in a subject, particularly for re-proliferation of a specific cell type for a specified window of time after treatment ( repopulation), recycling, regeneration and/or expansion can be used to create a preferred state. Exemplary cell types include T cells, B cells, dendritic cells and other cells of the immune system.

T cell activation and expansion

Before or after genetic modification of T cells with a chimeric receptor, multispecific chimeric receptor or dual chimeric receptor system described herein, the T cells can be, for example, U.S. Patent number. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; And U.S. Patent application publication number. It is generally activated and can be extended using the methods described in 20060121005.

In general, T cells can be expanded by contact with a surface to which an agent that stimulates the CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the T cell surface is attached. In particular, the T cell population is as described herein, for example by contact with an anti-CD3 antibody or antigen binding fragment thereof, or by contact with an anti-CD2 antibody immobilized on the surface, or by calcium iono It can be stimulated by contact with a protein kinase C activator (eg, bryostatin) with the pore. For co-stimulation of an accessory molecule on the surface of a T cell, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable to stimulate the proliferation of T cells. To stimulate the proliferation of CD4+ T cells or CD8+ T cells, anti-CD3 antibody and anti-CD28 antibody. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), and can be used as in other methods commonly known in the art (Berg et al., Transplant Pr° C. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63 , 1999).

In some embodiments, the primary stimulation signal and co-stimulation signal for T cells may be provided by different protocols. For example, the agent providing each signal may be in solution or bound to a surface. When bound to a surface, the agents can be bound to the same surface (ie, to form a “cis”), or to separate surfaces (ie to form a “trans”). Alternatively, one agent may be bound to the surface and the other agent may be bound as a solution. In one embodiment, the agent providing the co-stimulatory signal is bound to the cell surface and the agent providing the primary activation signal is in solution or is bound to the surface. In certain embodiments, both agents may be in solution. In another embodiment, the agent may be in a soluble form and then cross-linked to a surface such as an antibody or other binding agent that will bind to the cell or agent expressing the Fc receptor. In this regard, for example, for artificial antigen-providing cells (aAPCs) considered to be used for T cell activation and expansion in the present invention, U.S. See patent application publication numbers 20040101519 and 20060034810.

In some embodiments, the T cells are combined with the agent-coated beads, and the beads and cells are subsequently separated before the cells are cultured. In an alternative embodiment, prior to cultivation, the agent-coated beads and cells are not separated, but are cultured together. In a further embodiment, the beads and cells are first enriched by application of a force such as a magnetic force, increasing the ligation of cell surface markers to induce cell stimulation.

For example, cell surface proteins can be ligated by bringing anti-CD3 and anti-CD28 attached paramagnetic beads (3 x 28 beads) into contact with T cells. In one embodiment, the cells (e.g., 10 ⁴ to 10 ⁹ T cells) and beads (e.g., DYNABEADS® M-450 CD3/CD28 T paramagnetic beads, 1:1 ratio) are buffered, preferably Is complexed in PBS (without divalent cations such as calcium and magnesium). Again, one of skill in the art can readily understand that any cell concentration can be used. For example, the target cells are very rare in the sample and may contain only 0.01% of the sample or the entire sample (ie, 100%) may contain the target cells of interest. Thus, any number of cells is within the scope of the present invention. In certain embodiments, in certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact of the cells and particles. have. For example, in one embodiment, a concentration of about 2 billion cells/ml is used. In another embodiment, more than 100 million cells/mL are used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells/mL is used. In still another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/mL is used. In additional embodiments, concentrations of 125 or 150 million cells/mL may be used. The use of high concentrations can increase cell yield, cell activation and cell expansion. In addition, the use of high cell concentrations makes it possible to more efficiently capture cells that can weakly express the target antigen of interest, such as CD28-negative T cells. In certain embodiments, such cell populations may have therapeutic value and would be desirable to obtain. For example, the use of high concentrations of cells generally allows more efficient selection of CD8+ T cells with weaker CD28 expression.

In some embodiments, the mixture can be incubated for several hours (about 3 hours) to about 14 days or any integer time value in between. In another embodiment, the mixture can be cultured for 21 days. In one embodiment of the invention, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desirable so that the incubation time of T cells can be at least 60 days. Conditions suitable for T cell culture include serum ( e.g. , fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL A suitable medium containing factors essential for proliferation and survival, including -12, IL-15, TGFβ, and TNF-α or any other additive for cell growth known to those skilled in the art ( e.g. , minimal essential medium or RPMI medium 1640 or X-vivo 15, (Lonza)). Other additives for cell growth include, but are not limited to, surfactants, plasmaates, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium contains serum-free, or an appropriate amount of serum (or in situ), or a specific set of hormones, and/or an appropriate amount of serum (or plasma) and/or an amount of cytokine(s) sufficient for growth and expansion of T cells. Optimizer with supplemented RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, additional amino acids, sodium pyruvate and vitamins. Antibiotics such as penicillin and streptomycin are included only in the experimental culture and not in the cell culture to be injected into the subject. The target cells are maintained under conditions necessary to support growth, eg, an appropriate temperature (eg, 37° C.) and atmosphere (eg, air+5% CO ₂ ). T cells exposed to various stimulation times can exhibit different properties. For example, a typical blood or apheresed peripheral blood mononuclear cell product has a larger helper T cell population (TH, CD4+) than the cytotoxic or inhibitor T cell population (TC, CD8). By stimulating the CD3 and CD28 receptors, the ex vivo expansion of T cells produces a T cell population about 8 to 9 days ago, mainly composed of TH cells, whereas after about 8 to 9 days, the T cell population is increasingly It contains a large population of TC cells. Therefore, depending on the therapeutic purpose, it may be advantageous to inject a T cell population mainly comprising TH cells into a subject. Similarly, if the antigen-specific subset of TC cells is isolated, it may be advantageous to expand this subset to a greater extent.

In addition, in addition to the CD4 and CD8 markers, other phenotypic markers change significantly, but largely, reproducibly during the cell expansion process. Thus, this reproducibility enables the ability to tailor activated T cell products for specific purposes.

IV. Pharmaceutical composition

Pharmaceutical compositions comprising any one of engineered immune effector cells comprising any one of the chimeric receptor, multispecific chimeric receptor or dual chimeric receptor systems described herein and a pharmaceutically acceptable carrier are disclosed herein More is provided on. Pharmaceutical compositions may contain selective pharmaceutically acceptable carriers, excipients or stabilizers for a number of engineered immune effector cells described herein having the desired purity in the form of lyophilized formulations or aqueous solutions (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed and are buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; Preservatives, tonicity agents, stabilizers, metal complexes (eg, Zn-protein complexes); Chelating agents such as EDTA and/or non-ionic surfactants.

Buffers are used to control the pH in a range that optimizes the therapeutic effect, especially when stability is pH dependent. The buffering agent is preferably present in a concentration ranging from about 50 mM to about 250 mM. Buffering agents suitable for use in the present invention include both organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffering agents may include histidine and trimethylamine salts such as tris.

Preservatives are added to retard microbial growth and are typically present in the range of 0.2% -1.0% (w/v). Preservatives suitable for use in the present invention include octadecyldimethylbenzyl ammonium chloride; Hexamethonium chloride; Benzalkonium halides (eg, chloride, bromide, iodide), benzethonium chloride; Thimerosal, phenol, butyl or benzyl alcohol; Alkyl parabens such as methyl or propyl parabens; Catechol; Resorcinol; Cyclohexanol, 3-pentanol and m-cresol.

There are tonicity agents sometimes known as "stabilizers" to adjust or maintain the tonicity of the liquid in the composition. When used with large charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with charged groups of amino acid side chains, thereby reducing the likelihood of inter- and intra-molecular interactions. Let it. The tonicity agent may be present in any amount of 0.1% to 25% by weight, preferably 1% to 5% by weight, taking into account the relative amounts of other ingredients. Preferred isotonic agents include polyhydric sugar alcohols, preferably trihydric or higher higher alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

Additional excipients include formulations that can provide one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) preventing denaturation or adhesion to container walls. Formulation. Such excipients include: polyhydric sugar alcohols (listed above); Amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like; Organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinicitose, myoinisitol, galactose, galactitol, glycerol , Cyclotol (eg, inositol), polyethylene glycol; Sulfur-containing reducing agents such as urea, glutathione, thioxic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; Low-molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Monosaccharides ( such as xylose, mannose, fructose, glucose; disaccharides ( such as lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

Non-ionic surfactants or detergents (also referred to as "wetting agents") are present to solubilize the therapeutic agent and protect the therapeutic protein from agitation-induced aggregation, which also causes denaturation of the active therapeutic protein or antibody. Without causing the formulation to be exposed to shear surface stress. The non-ionic surfactant is present in a range of about 0.05 mg/mL to about 1.0 mg/mL, preferably about 0.07 mg/mL to about 0.2 mg/mL.

Suitable non-ionic surfactants are polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers. (TWEEN®20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl cellulose And carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

In order for the pharmaceutical composition to be used for in vivo administration, it must be sterilized. The pharmaceutical composition may be sterilized by filtration through a sterile filtration membrane. The pharmaceutical composition is generally placed in a container having a sterile access port, for example an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

The route of administration is followed by known and accepted methods, e.g. single or multiple bolus or long-term infusion in an appropriate manner, such as subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, lesion By injection or infusion by the intraoral or intraarterial route, topical administration, by injection or infusion by inhalation or sustained release or extended-release means.

The pharmaceutical compositions described herein may also contain one or more active compounds or agents necessary for the particular indication being treated, preferably active compounds having complementary activities that do not adversely affect each other. Alternatively, or additionally, the composition may comprise a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, or a growth inhibitory agent. These molecules are present in an appropriate combination in an amount effective for the desired purpose.

V. How to Treat Cancer

The present application further relates to methods and compositions for use in cellular immunotherapy. In some embodiments, the cellular immunotherapy is for treating cancer including, but not limited to, hematologic malignancies and solid tumors. Any of the above chimeric receptors, multispecific chimeric receptors, dual chimeric receptor systems, and engineered immune effector cells (such as engineered T cells) described herein can be used in cancer treatment methods.

In some embodiments, a method of treating cancer (such as multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia) in a subject (such as a human subject) is provided, wherein the method comprises: It comprises administering an effective amount of a pharmaceutical composition comprising: (1) engineered immune effector cells comprising a chimeric receptor (such as T cells), wherein the chimeric receptor comprises: (a) the NKG2D domain Containing extracellular domain; (b) transmembrane domain; And (c) an intracellular signaling domain; And (2) a pharmaceutically acceptable carrier. In certain embodiments, the chimeric receptor comprises a polypeptide comprising, in the N-terminal to C-terminal direction, a first NKG2D domain, a second NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain, 4- The co-stimulatory signaling domain derived from 1BB, and the primary intracellular signaling domain derived from CD3ζ.

In some embodiments, a method of treating cancer (such as multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia) in a subject (such as a human subject) is provided, wherein the method comprises: And administering an effective amount of a pharmaceutical composition comprising: (1) the engineered immune effector cells (such as T cells) comprise a multispecific chimeric receptor comprising a polypeptide chain comprising: (a ) An extracellular domain comprising a first NKG2D domain, a second NKG2D domain, and a CD123 binding domain (eg, IL-3 domain); (b) transmembrane domain; And (c) an intracellular signaling domain; And (2) a pharmaceutically acceptable carrier. In some embodiments, the CD123 binding domain is an anti-CD123 antibody fragment ( eg , scFv or VHH). In some embodiments, the CD123 binding domain is an IL-3 domain. In some embodiments, the polypeptide chain in the N-terminal to C-terminal direction, comprises: an IL-3 domain, a first peptide linker, a first NKG2D domain, a second peptide linker, a second NKG2D domain, CD8α hinge region, CD8α transmembrane domain, co-stimulatory signaling domain derived from 4-1BB, and primary intracellular signaling domain derived from CD3ζ.

In some embodiments, a method of treating cancer (such as multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia) in a subject (such as a human subject) is provided, wherein the method comprises: And administering an effective amount of a pharmaceutical composition comprising: (1) engineered immune effector cells (such as T cells) comprising a chimeric receptor comprising a multispecific first polypeptide chain and a second polypeptide chain, , Wherein each chain comprises: (a) an extracellular domain including an NKG2D domain and a CD123 binding domain ( eg , an IL-3 domain); (b) transmembrane domain; And (c) an intracellular signaling domain; And (2) a pharmaceutically acceptable carrier. In some embodiments, the NKG2D domain of the first polypeptide chain is crosslinked to the NKG2D domain of the second polypeptide chain through one or more disulfide bonds. In some embodiments, each of the first polypeptide chain and the second polypeptide chain in the N-terminal to C-terminal direction comprises: an IL-3 domain, a peptide linker, an NKG2D domain, a CD8α hinge region, a CD8α transmembrane. Domain, a co-stimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the extracellular domain further comprises a dimerization motif ( eg , a leucine zipper or a cysteine zipper) disposed between the NKG2D domain and the CD123 binding domain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain in an N-terminal to C-terminal direction comprises: an IL-3 domain, a leucine zipper, a NKG2D domain, a CD8α hinge region, a CD8α transmembrane. Domain, a co-stimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ.

In some embodiments, a method of treating cancer (such as multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia) in a subject (such as a human subject) is provided, wherein the method comprises: And administering an effective amount of a pharmaceutical composition comprising: (1) the engineered immune effector cells (such as T cells) comprise a dual chimeric receptor system comprising: (i) a first polypeptide chain and Each chain of a first chimeric receptor comprising a second polypeptide chain comprises: (a) an extracellular domain comprising a first NKG2D domain; (b) a first transmembrane domain; And (c) a first intracellular signaling domain; And (ii) a second chimeric receptor comprising a third polypeptide chain comprising: (a) a second extracellular domain comprising a second antigen binding domain; (b) a second transmembrane domain; And optionally (c) a second intracellular signaling domain; And (2) a pharmaceutically acceptable carrier. In some embodiments, each of the first polypeptide chain and the second polypeptide chain in the N-terminal to C-terminal direction comprises: NKG2D domain, CD8α hinge region, CD8α transmembrane domain, derived from 4-1BB. A co-stimulatory signaling domain, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the second chimeric receptor comprises a polypeptide comprising, in the N-terminal to C-terminal direction, an IL-3 domain, a CD8α hinge region, a CD8α transmembrane domain, and optionally 4- Co-stimulatory signaling domain derived from 1BB.

The methods described herein are suitable for treating a variety of cancers, including both solid and liquid cancers. In certain embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia. In some embodiments, the cancer is refractory or relapsed cancer. The methods described herein include first therapy, second therapy, third therapy, or other types of cancer therapy known in the art, such as chemotherapy, surgery, radioactivity, gene therapy, immunotherapy, bone marrow transplantation, stem Cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radiofrequency ablation or the like can be used as a combination therapy in an adjuvant setting or a neoadjuvant setting.

Administration of the pharmaceutical composition can be carried out in any convenient manner, such as injection, transfusion, transplantation or transplantation. The composition may be administered to a patient intraarterial, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous or intraperitoneal. In some embodiments, the pharmaceutical composition is administered systemically. In certain embodiments, the pharmaceutical composition is administered to the subject by infusion, such as intravenous infusion. Infusion techniques for immunotherapy are known in the art ( eg , Rosenberg et al ., New Eng. J. of Med. 319: 1676 (1988)). In certain embodiments, the composition is administered by intravenous injection.

The dosage and desired drug concentration of the pharmaceutical composition of the present invention may vary depending on the specific application envisioned. Determination of an appropriate dosage or route of administration is within the skill of one of skill in the art. Animal testing provides a reliable guide for determining effective doses for human treatment. Interspecies scaling of effective doses can be performed according to the following principles: Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics”, In Toxicokinetics and New Drug Development , Yacobi et al . Eds, Pergamon Press, New York 1989, pp. 42-46. Different formulations will be effective for different treatments and different disorders, and administration to treat a particular organ or tissue is different from that to another organ or tissue. It is within the scope of this application that it may require delivery in a manner.

In some embodiments, the amount of pharmaceutical composition is effective to elicit a desired clinical response in the subject. In some embodiments, the amount of the pharmaceutical composition is effective to cause disease remission (partially or completely) in the subject. In some embodiments, the amount of the pharmaceutical composition is effective in preventing recurrence or progression of cancer in the subject. In certain embodiments, the amount of pharmaceutical composition is effective in prolonging survival (such as disease-free survival) in the subject. In some embodiments, the pharmaceutical composition is effective in improving the quality of life of the subject.

In some embodiments, the amount of the pharmaceutical composition is effective in inhibiting the growth or reducing the size of a solid or lymphoid tumor. In some embodiments, the size of the solid or lymphoid tumor is at least about 10% (e.g., at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, or any of 100% Including those of) are reduced.

In some embodiments, the amount of the pharmaceutical composition is effective to inhibit tumor metastasis in the subject. In some embodiments, at least about 10% (e.g., including any of at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) metastasis is inhibited. . In some embodiments, a method of inhibiting metastasis to a lymph node is provided. In some embodiments, a method of inhibiting metastasis to the lung is provided. Metastasis can be assessed by methods known in the art such as blood tests, bone scans, x-ray scans, CT scans, PET scans and biopsies.

VI. Kits and articles of manufacture

Kits, unit dosage forms and articles of manufacture comprising the chimeric receptor, multispecific chimeric receptor, dual chimeric receptor system, or the engineered immune effector cells described herein are further provided. In some embodiments, kits are provided containing any one of the pharmaceutical compositions described herein, preferably instructions for use thereof are provided.

The kit of the present application is suitably packaged. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretive information. The present application thus also provides an article of manufacture comprising vials (such as sealed vials), bottles, jars, flexible packaging and the like.

The article of manufacture may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed of various materials such as glass or plastic. In general, the container holds a composition effective for treating a disease or disorder (such as cancer) described herein, and may have a sterile access port (e.g., the container is by an intravenous solution bag or a hypodermic needle). It may be a vial with a pierceable stopper). The label or package insert indicates that the composition will be used to treat a particular condition in the individual. The label or package insert will further include instructions for administering the composition to the subject. Reconstitution and/or instructions for use may be indicated on the label. The container holding the pharmaceutical composition may be a multi-use vial, which allows repeated administration of the reconstituted formulation (eg, 2-6 administrations). Package insert refers to instructions customarily included in a commercial package of a therapeutic product, including information regarding indications, usage, dosage, administration, contraindications and/or warnings regarding the use of such a therapeutic product. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may further contain other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

The kit or article of manufacture may contain multiple unit doses and instructions for use of a pharmaceutical composition, for example, a pharmaceutical composition packaged in an amount sufficient for storage and use in hospital pharmacies and complex pharmacies.

Example

The examples below are intended to be purely illustrative of the invention and should not be regarded as limiting the invention in any way. The following examples and detailed descriptions are provided by way of example and not limitation.

Example 1. Preparation of NKG2D × IL-3 chimeric receptor and dual NKG2D/IL-3 chimeric receptor system

This example describes the planning and preparation of exemplary bispecific chimeric receptors, dual chimeric receptor systems, and engineered T cells.

Planning of bispecific chimeric receptor and dual chimeric receptor system structures

Tables 1 and 2 show the components of the seven constructs and corresponding sequences.

Structure 1: monomeric NKG2D × IL-3 chimeric receptor (LIC2001). The present bispecific chimeric receptor comprises a single polypeptide chain comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain in the N-terminal to C-terminal direction, wherein the extracellular domain is the N-terminal In the C-terminal direction, the IL-3 domain specific for CD123 binding, a first peptide linker, a first NKG2D domain having an arginine residue engineered at its N-terminus, a second peptide linker, and engineered at its C-terminus And a second NKG2D domain with an aspartic acid residue. The NKG2D domains are responsible for NKG2D ligand binding during dimerization. The engineered arginine residues and aspartic acid residues are associated with each other to prevent the binding of the NKG2D dimer to a free NKG2D ligand that does not bind to tumor cells. The nucleic acid construct encodes a single polypeptide chain.

Structure 2: monomeric NKG2D x IL-3 chimeric receptor (LIC2001-1). The present bispecific chimeric receptor comprises a single polypeptide chain comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain in the N-terminal to C-terminal direction, wherein the extracellular domain is the N-terminal In the C-terminal direction, an IL-3 domain specific for CD123 binding, a first peptide linker, a first NKG2D domain, a second peptide linker, and a second NKG2D domain. The NKG2D domains are responsible for NKG2D ligand binding during dimerization. The nucleic acid construct encodes a single polypeptide chain. For comparison purposes, a monospecific NKG2D chimeric receptor (LIC2001-2) was constructed, which in the N-terminal to C-terminal direction, comprising a first forward NKG2D domain, a peptide linker, and a second forward NKG2D domain. domain; Transmembrane domain (CD8α), and intracellular signaling domains (4-1BB and CD3ζ). The amino acid sequence of LIC2001-2 is SEQ ID NO: 33, and the nucleic acid sequence of LIC2001-2 is SEQ ID NO: 38.

Construct 3: Dimeric NKG2D x IL-3 chimeric receptor with leucine zipper motif (LIC2002). The present bispecific chimeric receptor comprises two identical polypeptide chains, wherein each chain comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain in the N-terminal to C-terminal direction, wherein the The extracellular domain comprises an IL-3 domain specific for CD123 binding, a leucine zipper motif, and a reverse NKG2D domain responsible for NKG2D ligand binding during homodimerization in the N-terminal to C-terminal direction. The nucleic acid construct encodes a single copy of the polypeptide chain.

Construct 4: Leucine zipper motif-free dimer NKG2D × IL-3 chimeric receptor (LIC2002-1). The present bispecific chimeric receptor comprises two identical polypeptide chains, wherein each chain comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain in the N-terminal to C-terminal direction, wherein the The extracellular domain comprises an IL-3 domain specific for CD123 binding, a peptide linker, and a reverse NKG2D domain responsible for NKG2D ligand binding during homodimerization in the N-terminal to C-terminal direction. The nucleic acid construct encodes a single copy of the polypeptide chain.

Construct 5: Dimeric NKG2D x IL-3 chimeric receptor with leucine zipper motif (LIC2002-2). The present bispecific chimeric receptor comprises two identical polypeptide chains, wherein each chain comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain in the N-terminal to C-terminal direction, wherein the The extracellular domain comprises an IL-3 domain specific for CD123 binding, a leucine zipper motif, and a forward NKG2D domain responsible for NKG2D ligand binding during homodimerization in the N-terminal to C-terminal direction. The nucleic acid construct encodes a single copy of the polypeptide chain.

Construct 6: Dual NKG2D/IL-3 chimeric receptor system (LIC2003). A polycistronic nucleic acid construct is designed for a dual NKG2D/IL-3 chimeric receptor system. The construct is in the N-terminal to C-terminal direction, the first polypeptide comprising the following (the extracellular domain is a reverse NKG2D domain and a forward NKG2D domain responsible for NKG2D ligand binding when homodimerization, a transmembrane domain and intracellular Signaling domain, followed by a T2A self-cleaving peptide) and a second polypeptide (IL-3 domain specific for CD123 binding followed by an intracellular signaling domain) comprising, in the N-terminal to C-terminal direction, , Including transmembrane domains). Upon expression of the construct, the first polypeptide forms a first chimeric receptor and the second polypeptide forms a second chimeric receptor.

Construct 7: Dual NKG2D/IL-3 chimeric receptor system (LIC2004). A polycistronic nucleic acid construct is designed for a dual NKG2D/IL-3 chimeric receptor system. The construct is in the N-terminal to C-terminal direction, the first polypeptide comprising the following (the extracellular domain is a forward NKG2D domain responsible for binding NKG2D ligand when homodimerization, a transmembrane domain and an intracellular signaling domain, T2A self-cleaving peptide) and a second polypeptide (IL-3 domain specific for CD123 binding, followed by an intracellular signaling domain, without a transmembrane domain) in the N-terminal to C-terminal direction, comprising: Inclusive). Upon expression of the construct, two copies of the first polypeptide form a first chimeric receptor, which is a dimer, and the second polypeptide forms a second chimeric receptor. A monospecific NKG2D chimeric receptor (LIC2004-1) is constructed, which in the N-terminal to C-terminal direction, an extracellular domain including a forward NKG2D domain, a CD8α hinge domain; Transmembrane domain (CD8α), and intracellular signaling domains (4-1BB and CD3ζ). The amino acid sequence of LIC2004-1 is SEQ ID NO: 35, and the nucleic acid sequence of LIC2004-1 is SEQ ID NO: 40.

Generation of lentiviral expression vectors

Briefly, the lentiviral vector is modified by replacing the original promoter with the human elongation factor 1α promoter (hEF1α) gene, with EcoR I and Xba I (GenScript) using pLVX-Puro (Clontech#632164). . The NKG2D × IL-3 chimeric receptor gene or the NKG2D/IL-3 dual chimeric receptor system gene is constructed by GenScript, and is cloned into the vector through EcoR I/ Hpa I to provide a recombinant lentiviral expression plasmid, which is a lentivirus It goes through the packaging process further.

The lentiviral packaging plasmid mixture containing pMDLg/pRRE (Addgene#12251), pRSV-Rev (Addgene#12253), and pMD2.G (Addgene#12259) was combined with polyetherimide (PEI), pLVX-NKG2D × IL-3 chimeric receptor/dual chimeric receptor system-Puro expression plasmids are pre-mixed in a pre-optimized ratio, mixed appropriately, and incubated for 5 minutes at room temperature. Then, the transfection mixture was added dropwise to the 293FT cells and mixed gently. The cells were then incubated overnight in a 37° C. and 5% CO ₂ cell incubator. The supernatant was collected after centrifuging at 4° C. 500g for 10 minutes. After the supernatant was filtered through a 0.45 μm PES filter, the virus supernatant was concentrated by 20% sucrose gradient ultracentrifugation. After centrifugation, the supernatant was carefully discarded and the virus pellet was carefully rinsed with pre-cooled DPBS. And the concentration of the virus was measured. Viruses were properly aliquoted and stored immediately at -80°C. The viral titer was determined by p24 based on the HTRF kit developed by GenScript. The following recombinant lentiviral expression plasmids were prepared to correspond to each of the seven constructs described above: pLLV-LIC2001, pLLV-LIC2001-1, pLLV-LIC2002, pLLV-LIC2002-1, pLLV-LIC2002-2, pLLV-LIC2003 , And pLLV-LIC2004, as well as pLLV-LIC2001-2 and pLLV-LIC2004-1.

PBMC preparation

Leukocytes were collected and the cell concentration was adjusted to 5×10 ⁶ cells/mL in R10 medium. The leukocytes were then mixed with 0.9% NaCl solution at a 1:1 (v/v) ratio. 3 mL lymphoprep medium was added to a 15 mL centrifuge tube, and 6 mL of the diluted lymphocyte mixture was slowly stacked on top of the lymph preparation. The lymphocyte mixture was centrifuged for 30 minutes at 800 g at 20° C. without brakes. The lymphocyte buffy coat was then collected with a 200 μL pipette. The collected fractions were diluted at least 6 times with 0.9% NaCl or R10 and the density of the solution was reduced. The harvested fraction was then centrifuged at 250 g for 10 minutes at 20°C. The supernatant was completely aspirated and 10 mL of R10 was added to the cell pellet. The mixture was further centrifuged at 20° C. at 250 g for 10 minutes. Subsequently, the supernatant was aspirated. R10, preheated to 2 mL 37[deg.] C., with 100 IU/mL IL-2 was added to the cell pellet, and the cell pellet was gently resuspended. Then the number of cells was counted, and PBMC samples were prepared for later experiments.

T cell purification

Purified from human T cell PBMCs using the Miltenyi Pan T cell isolation kit (Cat#130-096-535) according to the protocol provided by the manufacturer as follows. The cell number was first measured. The cell suspension was centrifuged at 300 g for 10 minutes. The supernatant was completely aspirated, and the cell pellet was resuspended in 40 μL buffer per total 10 ⁷ total cells. 10 μL of Pan T cell biotin-antibody cocktail was added per 10 ⁷ total cells, mixed thoroughly, and incubated for about 5 minutes in a refrigerator (2-8° C.). Then 30 μL of buffer was added 10 ⁷ per cell. 20 μL of Pan T Cell MicroBead cocktail was added per 10 ⁷ cells. The mixture was mixed well and incubated for an additional 10 minutes in a refrigerator (2-8° C.). At least 500 μL is required for magnetic field separation. The LS column was placed in the magnetic field of a suitable MACS separator. The column was prepared by rinsing with 3 mL of buffer. The cell suspension is then applied onto the column and the flow-through containing unlabeled cells is collected to reveal the concentrated T cell fraction. The column is then washed with 3 mL of buffer to collect T cells, and unlabeled cells passing through are collected, representing concentrated T cells, which bind with the pass-through from the previous step. T cells were then resuspended in R10+100IU/mL IL-2. The primary T cells were then pre-activated with a human T cell activation/extension kit (Miltenyi #130-091-441) for 3 days, prior to transduction.

Purified T cells were transfected with each of the recombinant lentiviral vectors, or the mRNA encoding each of the chimeric receptor constructs was transfected via electroporation, and then incubated overnight in a 37°C, 5% CO ₂ incubator. . Engineered T cells expressing each of the seven constructs described in this example were prepared.

Expression of chimeric receptors on engineered T cells

The mRNA molecules encoding the LIC2002-2 and LIC2004 chimeric receptor constructs were delivered to T cells, respectively, by electroporation. Expression of the chimeric receptor was detected using a flow cytometric assay. Briefly, electroporated T cells were harvested, rinsed with DPBS, and then containing 2 μL of PE-conjugated CD314 protein (MILTENYI BIOTEC, 130-111-645) for NKG2D domain detection, or Resuspended in 100 μL DPBS containing 10 μL of PE-conjugated anti-IL-3 antibody (MILTENYI BIOTEC, 130-096-084) for IL-3 domain detection. The reaction mixture was incubated at 4° C. for 20 minutes. Later, cells were washed with 200 μL DPBS, resuspended in DPBS, and analyzed by flow cytometry. As shown in Figure 2, the engineered T cells express chimeric receptor(s) having NKG2D domains and IL-3 domains.

In vitro Cytotoxicity assay

The engineered T cells were harvested and seeded into 384-well reaction plates. Target cells were human chronic myelogenous leukemia (CML) cell lines K562-Luc and K562-CD123-Luc that recombinantly express CD123. All cell lines were engineered in-house to express firefly luciferase. To assay the cytotoxicity of engineered T cells against tumor cells, the engineered T cells were subjected to the target cells at the effector (engineered T cells) ratio (“E:T ratio”) to target cells for 20 hours. It was co-incubated with the cells. ONE-GLO ^TM Luminescent Luciferase Assay Reagent (Promega # E6110) was prepared according to the manufacturer's protocol, and added to co-cultured cells to detect the remaining luciferase activity in each well. Since luciferase was expressed only in target cells, the luciferase activity remaining in the well was directly related to the number of viable target cells in that well. Maximum luciferase activity was obtained by adding culture medium to target cells in the absence of effector cells. Minimum luciferase activity was determined by adding Triton X-100 as a positive control at a final concentration of 1%. Specific lysis/cytotoxicity was calculated according to the following equation:

Specific lysis/cytotoxicity%=100%×[1-(LUC sample-LUCminimum)/(LUCmaximum-LUCminimum)]. The luciferase value (“LUC” or luminescence) is proportional to the amount of viable cells in each reaction well.

As shown in Fig. 3A, T cells expressing LIC2001-1 (76.63±4.58%), T cells expressing LIC2002-2 (96.52±1.68%) and T cells expressing LIC2004 (96.89±0.70%) were It demonstrates a significant killing effect against K562-CD123-Luc. As shown in FIG. 3B, a killing effect was also observed for K562-Luc cells: LIC2001-1 (35.98±6.08%) expressing T cells, LIC2002-2 expressing T cells (94.42±2.66%), and LIC2004 (95.87±1.61%) expressing T cells. However, the engineered T cells expressing various NKG2D × IL-3 chimeric receptor constructs showed higher cytotoxicity against K562-CD123-Luc cells than K562-Luc cells.

T cells expressing LIC2002-2 and T cells expressing LIC2004 were subjected to an effector (engineered T cell) ratio to target cells of 10:1, 5: in order to evaluate cytotoxicity in vitro against KG1-Luc. Co-incubated with the human acute myelogenous leukemia (AML) cell line KG1-Luc at 1 or 2.5:1. As shown in Fig. 3C, T cells expressing LIC2002-2 (83.68±7%, 78.55±4%, and 60.87±10%) and T cells expressing LIC2004 (85.6±3%, 76.89±4%,) And 75.53±1%) strong and dose-dependent cytotoxic effects were observed.

The cytotoxic activity of T expressing LIC2002-2, T expressing LIC2004 and T expressing LIC2004-1 against K562-CD123-Luc at various effector: target ratios was compared. The LIC2004-1 construct is the NKG2D chimeric receptor in the LIC2004 dual chimeric receptor system. These results are shown in FIG. 4. The Y-axis represents the percentage of specific killing, and the X-axis represents the natural logarithm of the effector: target ratio ( eg , engineered T cells: K562-CD123-Luc cells). The logarithm of the E:T ratio is plotted against the percentage of specific deaths fitted to the dotted line by linear regression. The smaller the slope of the fitted line, the stronger the killing ability. This result demonstrates that the LIC2004 dual chimeric receptor system has higher efficacy than the corresponding NKG2D chimeric receptor alone ( eg , LIC2004-1; p=0.031). There was no significant difference between the bispecific chimeric receptor constructs LIC2002-2 and LIC2004 (p=0.277). Statistical analysis was performed using Graphpad Prism 6.

Mechanism of action

A series of in vitro cytotoxicity assays were performed to study the mechanism of engineered T cells expressing the NKG2D x IL-3 chimeric receptor construct. First, “NKG2D-CD123 T cells” (LIC2004 expressing T cells), “NKG2D T cells” (T cells expressing NKG2D-CD8 hinge-CD8TM-4-1BB-CD3ζ), and “CD123 conjugate T cells” (T cells expressing IL3-CD8 hinge-CD8TM) were co-cultured with target K562-CD123-Luc and K562-Luc cells in turn at an E:T ratio of 20:1.

As shown in Figure 5a, NKG2D-CD123 T cells (70.59±1.5%) showed a significant tumor killing effect, while the tumor killing effect of NKG2D T cells (42.14±8.4%) was much weaker, and CD123 Conjugate T cells did not appear to be cytotoxic against target tumor cells. In Fig. 5b, NKG2D-CD123 T cells also showed significant cytotoxicity (88.28±6.58%) against K562-luc cells, while NKG2D T cells showed lower cytotoxicity (66.68±2.87%), and CD123 conjugate T cells showed no cytotoxicity (-46.98±11.22%) against K562-Luc cells. These results indicate that NKG2D-CD123 T cells are more effective than NKG2D T cells.

Moreover, cytotoxicity assays were run in the presence or absence of soluble MICA, a cognate ligand of NKG2D. NKG2D-CD123 T cells (LIC2002-2 or LIC2004), when co-cultured with the target cells (K562-Luc or K562-CD123-Luc) at an E:T ratio of 20:1, in turn were converted to recombinant MICA protein or BSA protein. Blocked. MICA or BSA was added to the co-culture at a concentration of 0 ng/mL, 100 ng/mL or 1000 ng/mL. T cells electroporated without mRNA served as a negative control.

As shown in FIGS. 6A-6B, treatment with MICA at the highest concentration tested was able to block the NKG2D domain and reduce the cytotoxic activity of T cells expressing LIC2002-2 and LIC2004. Treatment with non-specific BSA did not significantly affect the cytotoxicity of the engineered T cells against tumor cells.

As shown in Figure 7, 20:1, 10:1, 5:1, 2.5:1, 1.25 of effectors (engineered T cells) against T cells expressing LIC2002-2 and target cells expressing LIC2004. Co-cultured with the K562-CD123-Luc or K562-Luc cell line at a :1 or 0.625:1 ratio. This result shows the dose-dependent killing effect of LIC2004 and LIC2002-2 in this K562 cell line. A stronger killing effect was observed in K562 cell lines expressing both NKG2D and CD123 than in K562 cells expressing NKG2D alone.

IFNγ release

Engineered T cells expressing LIC2002-2, LIC2004 or LIC2004-1 constructs were co-cultured with the K562-CD123-Luc, K562-Luc, or KG1-Luc cell lines in turn for 20 h. Supernatants from co-cultures were collected and evaluated to determine levels of cytokine release ( eg , interferon gamma, IFNγ release).

Figure 8a shows the level of IFNγ in the cell-free supernatant after co-culture of engineered T cells with CD123 positive K562-CD123-Luc for 20 hours. Secreted IFNγ levels were 1526.51±92.13 pg/mL (LIC2002-2), 1089.36±8.06 pg/mL (LIC2004), 687.62±31.65 pg/mL (LIC2004-1), and 64.15±16.56 pg/mL (control without RNA). ). IFNγ was undetectable in the control without T cells.

Figure 8b shows the level of IFNγ in the cell-free supernatant after co-culture of engineered T cells with K562-Luc for 20 hours. Secreted IFNγ levels were 3416.67±71.15 pg/mL (LIC2002-2), 3063.46±119.46 pg/mL (LIC2004), 1841.41±222.18 pg/mL (LIC2004-1), and 3.99±11.57 pg/mL (control without RNA). ). IFNγ was undetectable in the control without T cells.

Figure 8c shows the level of IFNγ in the cell-free supernatant after co-culture of engineered T cells with KG1-Luc for 20 hours. Secreted IFNγ levels were 267.75±34.33 pg/mL (LIC2002-2), 265.87±12.19 pg/mL (LIC2004), 236.56±16.45 pg/mL (LIC2004-1), and 220.56±80.5 pg/mL (control without RNA). ). IFNγ was undetectable in the control without T cells.

SEQUENCE LISTING <110> NANJING LEGEND BIOTECH CO.,Ltd Fan, Xiaohu Wang, Jun Wang, Pingyan Zhuang, Qiuchuan Ma, Lian <120> MULTISPECIFIC CHIMERIC RECEPTORS COMPRISING AN NKG2D DOMAIN AND METHODS OF USE THEREOF <130> 76142-20009.41 <140> Not Yet Assigned <141> Concurrently Herewith <150> PCT/CN2017/119397 <151> 2017-12-28 <160> 45 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 1 Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gln Leu Leu Val Arg Pro 1 5 10 15 Gly Leu Gln <210> 2 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 2 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 3 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 3 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 4 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 4 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 5 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 5 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 6 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 6 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 7 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 7 Val Thr Arg Gln Met Cys Ile Tyr Thr Asn Pro Thr Ser Cys Asn Glu 1 5 10 15 Ile Tyr Gly Lys Phe Ser Ser Ala Tyr Leu Ala Cys Asp Gly Lys Gln 20 25 30 Met Glu Ile Ile Thr Leu Leu Asn Pro Ser Leu Ile Ser Gly Asp Glu 35 40 45 Trp Gln Trp Ser Gly Asn Thr Pro Ile His Val Leu Gly Met Trp His 50 55 60 Tyr Ser Lys Val Leu Lys Leu Leu Asp Gln Asp Glu Lys Ser Tyr Val 65 70 75 80 Lys Leu Leu Ser Ala Asn Gln Ser Met Cys Ser Ala Gln Ser Glu Tyr 85 90 95 Trp Asn Lys Ser Glu Asp Phe Phe Gln Tyr Cys Asn Asn Lys Tyr Cys 100 105 110 Ile Trp Asn Lys Pro Cys Pro Gly Cys Tyr Ser Glu Thr Leu Pro Ile 115 120 125 <210> 8 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 8 Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile 1 5 10 15 Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp 20 25 30 Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys 35 40 45 Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr 50 55 60 His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp 65 70 75 80 Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met 85 90 95 Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile 100 105 110 Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val 115 120 125 <210> 9 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 9 Ala Pro Met Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp Val Asn Cys 1 5 10 15 Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu 20 25 30 Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu 35 40 45 Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala 50 55 60 Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn 65 70 75 80 Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro 85 90 95 Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr 100 105 110 Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu 115 120 125 Ser Leu Ala Ile Phe 130 <210> 10 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 10 Lys Glu Glu Leu Glu Ala Glu Lys Arg Asp Leu Ile Arg Thr Asn Glu 1 5 10 15 Arg Leu Ser Gln Glu Leu Glu Tyr Leu 20 25 <210> 11 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 11 Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu 1 5 10 15 Glu Asn Pro Gly Pro 20 <210> 12 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 12 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser 20 <210> 13 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 13 Ser Gly Gly Gly Gly Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly 20 <210> 14 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 14 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Ser Gly Gly Gly Gly Ser 20 <210> 15 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly 1 5 10 15 Ser Gly Ser Gly Gly Gly Gly Ser 20 <210> 16 <211> 704 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 16 Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gln Leu Leu Val Arg Pro 1 5 10 15 Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp 20 25 30 Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln 35 40 45 Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln 50 55 60 Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe 65 70 75 80 Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile 85 90 95 Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr 100 105 110 Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg 115 120 125 Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 130 135 140 Thr Thr Leu Ser Leu Ala Ile Phe Thr Ser Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Gly Gly Gly Ser 165 170 175 Arg Arg Val Thr Arg Gln Met Cys Ile Tyr Thr Asn Pro Thr Ser Cys 180 185 190 Asn Glu Ile Tyr Gly Lys Phe Ser Ser Ala Tyr Leu Ala Cys Asp Gly 195 200 205 Lys Gln Met Glu Ile Ile Thr Leu Leu Asn Pro Ser Leu Ile Ser Gly 210 215 220 Asp Glu Trp Gln Trp Ser Gly Asn Thr Pro Ile His Val Leu Gly Met 225 230 235 240 Trp His Tyr Ser Lys Val Leu Lys Leu Leu Asp Gln Asp Glu Lys Ser 245 250 255 Tyr Val Lys Leu Leu Ser Ala Asn Gln Ser Met Cys Ser Ala Gln Ser 260 265 270 Glu Tyr Trp Asn Lys Ser Glu Asp Phe Phe Gln Tyr Cys Asn Asn Lys 275 280 285 Tyr Cys Ile Trp Asn Lys Pro Cys Pro Gly Cys Tyr Ser Glu Thr Leu 290 295 300 Pro Ile Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 305 310 315 320 Gly Ser Gly Ser Gly Gly Gly Gly Ser Ile Pro Leu Thr Glu Ser Tyr 325 330 335 Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr 340 345 350 Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys 355 360 365 Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln 370 375 380 Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His 385 390 395 400 Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser 405 410 415 Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu 420 425 430 Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn 435 440 445 Thr Tyr Ile Cys Met Gln Arg Thr Val Asp Asp Ser Gly Gly Gly Gly 450 455 460 Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 465 470 475 480 Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 485 490 495 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 500 505 510 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 515 520 525 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 530 535 540 Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 545 550 555 560 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 565 570 575 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 580 585 590 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 595 600 605 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 610 615 620 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 625 630 635 640 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 645 650 655 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 660 665 670 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 675 680 685 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 690 695 700 <210> 17 <211> 700 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 17 Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gln Leu Leu Val Arg Pro 1 5 10 15 Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp 20 25 30 Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln 35 40 45 Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln 50 55 60 Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe 65 70 75 80 Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile 85 90 95 Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr 100 105 110 Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg 115 120 125 Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 130 135 140 Thr Thr Leu Ser Leu Ala Ile Phe Thr Ser Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Gly Gly Gly Ser 165 170 175 Val Thr Arg Gln Met Cys Ile Tyr Thr Asn Pro Thr Ser Cys Asn Glu 180 185 190 Ile Tyr Gly Lys Phe Ser Ser Ala Tyr Leu Ala Cys Asp Gly Lys Gln 195 200 205 Met Glu Ile Ile Thr Leu Leu Asn Pro Ser Leu Ile Ser Gly Asp Glu 210 215 220 Trp Gln Trp Ser Gly Asn Thr Pro Ile His Val Leu Gly Met Trp His 225 230 235 240 Tyr Ser Lys Val Leu Lys Leu Leu Asp Gln Asp Glu Lys Ser Tyr Val 245 250 255 Lys Leu Leu Ser Ala Asn Gln Ser Met Cys Ser Ala Gln Ser Glu Tyr 260 265 270 Trp Asn Lys Ser Glu Asp Phe Phe Gln Tyr Cys Asn Asn Lys Tyr Cys 275 280 285 Ile Trp Asn Lys Pro Cys Pro Gly Cys Tyr Ser Glu Thr Leu Pro Ile 290 295 300 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 305 310 315 320 Gly Ser Gly Gly Gly Gly Ser Ile Pro Leu Thr Glu Ser Tyr Cys Gly 325 330 335 Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe 340 345 350 Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser 355 360 365 Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu 370 375 380 Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro 385 390 395 400 Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn 405 410 415 Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala 420 425 430 Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr 435 440 445 Ile Cys Met Gln Arg Thr Val Ser Gly Gly Gly Gly Ser Gly Ser Gly 450 455 460 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Thr Thr Thr 465 470 475 480 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 485 490 495 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 500 505 510 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 515 520 525 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 530 535 540 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 545 550 555 560 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 565 570 575 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 580 585 590 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 595 600 605 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 610 615 620 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 625 630 635 640 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 645 650 655 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 660 665 670 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 675 680 685 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 690 695 700 <210> 18 <211> 552 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 18 Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gln Leu Leu Val Arg Pro 1 5 10 15 Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp 20 25 30 Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln 35 40 45 Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln 50 55 60 Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe 65 70 75 80 Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile 85 90 95 Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr 100 105 110 Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg 115 120 125 Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 130 135 140 Thr Thr Leu Ser Leu Ala Ile Phe Thr Ser Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Gly Ser Gly Gly Gly 165 170 175 Gly Ser Lys Glu Glu Leu Glu Ala Glu Lys Arg Asp Leu Ile Arg Thr 180 185 190 Asn Glu Arg Leu Ser Gln Glu Leu Glu Tyr Leu Val Thr Arg Gln Met 195 200 205 Cys Ile Tyr Thr Asn Pro Thr Ser Cys Asn Glu Ile Tyr Gly Lys Phe 210 215 220 Ser Ser Ala Tyr Leu Ala Cys Asp Gly Lys Gln Met Glu Ile Ile Thr 225 230 235 240 Leu Leu Asn Pro Ser Leu Ile Ser Gly Asp Glu Trp Gln Trp Ser Gly 245 250 255 Asn Thr Pro Ile His Val Leu Gly Met Trp His Tyr Ser Lys Val Leu 260 265 270 Lys Leu Leu Asp Gln Asp Glu Lys Ser Tyr Val Lys Leu Leu Ser Ala 275 280 285 Asn Gln Ser Met Cys Ser Ala Gln Ser Glu Tyr Trp Asn Lys Ser Glu 290 295 300 Asp Phe Phe Gln Tyr Cys Asn Asn Lys Tyr Cys Ile Trp Asn Lys Pro 305 310 315 320 Cys Pro Gly Cys Tyr Ser Glu Thr Leu Thr Thr Thr Pro Ala Pro Arg 325 330 335 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 340 345 350 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 355 360 365 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 370 375 380 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg 385 390 395 400 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 405 410 415 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 420 425 430 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 435 440 445 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 450 455 460 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 465 470 475 480 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 485 490 495 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 500 505 510 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 515 520 525 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 530 535 540 His Met Gln Ala Leu Pro Pro Arg 545 550 <210> 19 <211> 527 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 19 Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gln Leu Leu Val Arg Pro 1 5 10 15 Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp 20 25 30 Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln 35 40 45 Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln 50 55 60 Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe 65 70 75 80 Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile 85 90 95 Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr 100 105 110 Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg 115 120 125 Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 130 135 140 Thr Thr Leu Ser Leu Ala Ile Phe Thr Ser Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Gly Ser Gly Gly Gly 165 170 175 Gly Ser Val Thr Arg Gln Met Cys Ile Tyr Thr Asn Pro Thr Ser Cys 180 185 190 Asn Glu Ile Tyr Gly Lys Phe Ser Ser Ala Tyr Leu Ala Cys Asp Gly 195 200 205 Lys Gln Met Glu Ile Ile Thr Leu Leu Asn Pro Ser Leu Ile Ser Gly 210 215 220 Asp Glu Trp Gln Trp Ser Gly Asn Thr Pro Ile His Val Leu Gly Met 225 230 235 240 Trp His Tyr Ser Lys Val Leu Lys Leu Leu Asp Gln Asp Glu Lys Ser 245 250 255 Tyr Val Lys Leu Leu Ser Ala Asn Gln Ser Met Cys Ser Ala Gln Ser 260 265 270 Glu Tyr Trp Asn Lys Ser Glu Asp Phe Phe Gln Tyr Cys Asn Asn Lys 275 280 285 Tyr Cys Ile Trp Asn Lys Pro Cys Pro Gly Cys Tyr Ser Glu Thr Leu 290 295 300 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 305 310 315 320 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 325 330 335 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 340 345 350 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 355 360 365 Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 370 375 380 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 385 390 395 400 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg 405 410 415 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 420 425 430 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 435 440 445 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 450 455 460 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 465 470 475 480 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 485 490 495 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 500 505 510 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 515 520 525 <210> 20 <211> 554 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 20 Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gln Leu Leu Val Arg Pro 1 5 10 15 Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp 20 25 30 Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln 35 40 45 Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln 50 55 60 Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe 65 70 75 80 Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile 85 90 95 Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr 100 105 110 Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg 115 120 125 Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 130 135 140 Thr Thr Leu Ser Leu Ala Ile Phe Thr Ser Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Gly Ser Gly Gly Gly 165 170 175 Gly Ser Lys Glu Glu Leu Glu Ala Glu Lys Arg Asp Leu Ile Arg Thr 180 185 190 Asn Glu Arg Leu Ser Gln Glu Leu Glu Tyr Leu Ile Pro Leu Thr Glu 195 200 205 Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn 210 215 220 Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala 225 230 235 240 Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu 245 250 255 Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu 260 265 270 Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile 275 280 285 Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys 290 295 300 Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr 305 310 315 320 Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro Ala 325 330 335 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 340 345 350 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 355 360 365 Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 370 375 380 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 385 390 395 400 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 405 410 415 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 420 425 430 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 435 440 445 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 450 455 460 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 465 470 475 480 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 485 490 495 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 500 505 510 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 515 520 525 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 530 535 540 Ala Leu His Met Gln Ala Leu Pro Pro Arg 545 550 <210> 21 <211> 2112 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 21 atgagtagac tgcccgtgct gctgctgctg cagctgctgg tgcgccccgg actgcaggcc 60 ccaatgaccc agacaacacc tctgaaaacc tcttgggtga actgcagcaa tatgatcgac 120 gagatcatca cacacctgaa gcagccccct ctgccactgc tggatttcaa caatctgaac 180 ggcgaggacc aggatatcct gatggagaac aatctgagac ggcccaacct ggaggccttt 240 aatagggccg tgaagagcct gcagaacgcc agcgccatcg agtccatcct gaagaatctg 300 ctgccatgtc tgcctctggc aaccgcagca ccaacacgcc acccaatcca catcaaggac 360 ggcgattgga acgagttcag gcgcaagctg accttttacc tgaagacact ggagaatgcc 420 caggcccagc agaccacact gtccctggcc atcttcacct ccggcggcgg cggctctgga 480 ggaggaggaa gcggaggagg aggatctgga tctggcggag gaggctctcg gagagtgaca 540 cggcagatgt gcatctatac caaccccaca agctgtaatg agatctacgg caagtttagc 600 tccgcctatc tggcctgcga cggcaagcag atggagatca tcaccctgct gaacccttct 660 ctgatcagcg gcgatgagtg gcagtggtcc ggcaatacac caatccacgt gctgggcatg 720 tggcactact ctaaggtgct gaagctgctg gaccaggatg agaagtccta tgtgaagctg 780 ctgtctgcca accagtccat gtgctctgcc cagagcgagt actggaataa gagcgaggac 840 ttctttcagt actgcaacaa taagtattgt atctggaaca agccatgccc cggctgttat 900 tccgagacac tgcctatctc tggcggagga ggatccggcg gcggcggctc cggcggcgga 960 ggaagcggct ccggcggcgg cggcagcatc ccactgacag agtcctactg cggcccttgt 1020 ccaaagaatt ggatctgcta caagaacaac tgttaccagt tctttgatga gagcaagaac 1080 tggtatgagt cccaggcctc ttgcatgagc cagaatgcct ctctgctgaa ggtgtacagc 1140 aaggaggacc aggatctgct gaagctggtg aagagctatc actggatggg cctggtgcac 1200 atccccacca acggctcctg gcagtgggag gacggctcca tcctgtctcc taatctgctg 1260 acaatcatcg agatgcagaa gggcgattgt gccctgtacg cctctagctt caagggctat 1320 atcgagaact gctccacccc taatacatac atctgtatgc agcggaccgt ggacgattct 1380 ggcgggggag gcagtgggtc agggggaggg ggaagcggag gaggagggag cggcgggggg 1440 ggcaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 1500 ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgaggggg 1560 ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 1620 ctcctgtcac tggttatcac cctttactgc aaacggggca gaaagaaact cctgtatata 1680 ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc 1740 cgatttccag aagaagaaga aggaggatgt gaactgagag tgaagttcag caggagcgca 1800 gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1860 agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1920 ccgagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 1980 gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 2040 ctttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 2100 ctgccccctc gc 2112 <210> 22 <211> 2094 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 22 atgagccgac tgcccgtgct gctgctgctg cagctgctgg tgcgacccgg actgcaggcc 60 cccatgaccc agactacccc actgaaaacc tcttgggtga actgcagcaa tatgatcgac 120 gagatcatca cacacctgaa gcagccccct ctgccactgc tggatttcaa caatctgaac 180 ggcgaggacc aggatatcct gatggagaac aatctgagac ggcccaacct ggaggccttt 240 aatcgggccg tgaagagcct gcagaacgcc agcgccatcg agtccatcct gaagaatctg 300 ctgccatgtc tgcctctggc aaccgcagca ccaacaagac acccaatcca catcaaggac 360 ggcgattgga acgagttcag gcgcaagctg accttttacc tgaagacact ggagaatgcc 420 caggcccagc agaccacact gtccctggcc atcttcacct ccggcggcgg cggctctgga 480 ggaggaggaa gcggaggagg aggatctgga tctggcggag gaggctccgt gacaaggcag 540 atgtgcatct ataccaaccc cacatcttgt aatgagatct acggcaagtt tagctccgcc 600 tatctggcct gcgacggcaa gcagatggag atcatcaccc tgctgaaccc ttctctgatc 660 agcggcgatg agtggcagtg gagcggcaat acaccaatcc acgtgctggg catgtggcac 720 tactccaagg tgctgaagct gctggaccag gatgagaagt cctatgtgaa gctgctgtct 780 gccaaccagt ccatgtgctc tgcccagagc gagtactgga ataagtccga ggacttcttt 840 cagtactgca acaataagta ttgtatctgg aacaagccat gccccggctg ttattctgag 900 acactgccta tctctggcgg aggaggatcc ggcggcggcg gctccggcgg cggaggaagc 960 ggctccggcg gcggcggcag catcccactg acagagtcct actgcggccc ttgtccaaag 1020 aattggatct gctacaagaa caactgttac cagttctttg atgagagcaa gaactggtat 1080 gagtcccagg cctcttgcat gagccagaat gcctctctgc tgaaggtgta cagcaaggag 1140 gaccaggatc tgctgaagct ggtgaagtct tatcactgga tgggcctggt gcacatcccc 1200 accaacggca gctggcagtg ggaggacggc tccatcctgt ctcctaatct gctgacaatc 1260 atcgagatgc agaagggcga ttgtgccctg tacgcctcta gcttcaaggg ctatatcgag 1320 aactgctcca cccctaatac atacatctgt atgcagagaa ccgtgtctgg gggaggggga 1380 agcggaagtg gcggaggcgg ctctggcggg ggaggaagtg gagggaccac gacgccagcg 1440 ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag 1500 gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgcctgtgat 1560 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1620 accctttact gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1680 agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1740 gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtaccag 1800 cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1860 ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1920 caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1980 gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 2040 acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgc 2094 <210> 23 <211> 1656 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 23 atgagtagac tgcccgtgct gctgctgctg cagctgctgg tgcgccccgg actgcaggcc 60 ccaatgaccc agacaacccc actgaagacc tcttgggtga actgcagcaa tatgatcgac 120 gagatcatca cacacctgaa gcagccccct ctgccactgc tggatttcaa caatctgaac 180 ggcgaggacc aggatatcct gatggagaac aatctgagac ggcccaacct ggaggccttt 240 aatcgcgccg tgaagtctct gcagaacgcc agcgccatcg agtccatcct gaagaatctg 300 ctgccatgtc tgccactggc aaccgcagca cctacacggc acccaatcca catcaaggac 360 ggcgattgga acgagttcag gcgcaagctg accttttacc tgaagacact ggagaatgcc 420 caggcccagc agaccacact gagcctggcc atcttcacct ccggcggcgg cggctctgga 480 ggaggaggaa gcggcggagg aggaggaggc tctggcagcg gcggcggcgg ctctaaggag 540 gagctggagg ccgagaagcg ggacctgatc agaaccaatg agaggctgag ccaggagctg 600 gagtacctgg tgacacggca gatgtgcatc tataccaacc ctacatcctg taatgagatc 660 tacggcaagt ttagctccgc ctatctggcc tgcgacggca agcagatgga gatcatcacc 720 ctgctgaacc cctccctgat ctctggcgat gagtggcagt ggagcggcaa tacacctatc 780 cacgtgctgg gcatgtggca ctactccaag gtgctgaagc tgctggacca ggatgagaag 840 tcctatgtga agctgctgtc tgccaaccag agcatgtgct ccgcccagtc tgagtactgg 900 aataagtccg aggatttctt tcagtattgt aacaacaaat actgcatctg gaacaaaccc 960 tgtcccggct gctactcaga gaccctgacc acgacgccag cgccgcgacc accaacaccg 1020 gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 1080 gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 1140 ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg 1200 ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1260 caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1320 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 1380 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1440 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1500 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1560 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1620 tacgacgccc ttcacatgca ggccctgccc cctcgc 1656 <210> 24 <211> 1581 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 24 atgagtagac tgcccgtgct gctgctgctg cagctgctgg tgcgccccgg actgcaggcc 60 ccaatgaccc agacaacccc actgaaaacc tcttgggtga actgcagcaa tatgatcgac 120 gagatcatca cacacctgaa gcagccccct ctgccactgc tggatttcaa caatctgaac 180 ggcgaggacc aggatatcct gatggagaac aatctgagac ggcccaacct ggaggccttt 240 aatcgggccg tgaagtccct gcagaacgcc agcgccatcg agtccatcct gaagaatctg 300 ctgccatgtc tgccactggc aaccgcagca cctacaaggc acccaatcca catcaaggac 360 ggcgattgga acgagttcag gcgcaagctg accttttacc tgaagacact ggagaatgcc 420 caggcccagc agaccacact gtctctggcc atcttcacct ccggcggcgg cggctctgga 480 ggaggaggaa gcggcggagg aggaggaggc tctggcagcg gcggcggcgg cagcgtgaca 540 cggcagatgt gcatctatac caaccctaca tcctgtaatg agatctacgg caagtttagc 600 tccgcctatc tggcctgcga cggcaagcag atggagatca tcaccctgct gaacccctcc 660 ctgatctctg gcgatgagtg gcagtggtct ggcaatacac ctatccacgt gctgggcatg 720 tggcactaca gcaaggtgct gaagctgctg gaccaggatg agaagtccta tgtgaagctg 780 ctgtctgcca accagagcat gtgctccgcc cagtctgagt actggaataa gagcgaggac 840 ttctttcagt attgtaacaa caagtattgc atttggaaca agccctgccc cgggtgctat 900 tctgaaacac tgaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 960 tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 1020 acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 1080 ggggtccttc tcctgtcact ggttatcacc ctttactgca aacggggcag aaagaaactc 1140 ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1200 tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1260 aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 1320 ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1380 gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1440 aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1500 cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1560 atgcaggccc tgccccctcg c 1581 <210> 25 <211> 1662 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 25 atgagtagac tgcccgtgct gctgctgctg cagctgctgg tgcgacccgg cctgcaggct 60 ccaatgaccc agacaacccc actgaagacc tcttgggtga actgcagcaa tatgatcgac 120 gagatcatca cacacctgaa gcagccccct ctgcctctgc tggatttcaa caatctgaac 180 ggcgaggacc aggatatcct gatggagaac aatctgagac ggcccaacct ggaggccttt 240 aatcgcgccg tgaagagcct gcagaacgcc agcgccatcg agtccatcct gaagaatctg 300 ctgccttgtc tgccactggc aaccgcagca ccaacacggc accctatcca catcaaggac 360 ggcgattgga acgagttcag gcgcaagctg accttttacc tgaagacact ggagaatgcc 420 caggcccagc agaccacact gtccctggcc atcttcacct ccggcggcgg cggctctgga 480 ggaggaggaa gcggcggagg aggaggaggc tctggcagcg gcggcggcgg ctctaaggag 540 gagctggagg ccgagaagcg ggacctgatc agaaccaacg agaggctgag ccaggagctg 600 gagtacctga tccccctgac agagtcctat tgcggcccat gtcccaagaa ttggatctgc 660 tacaagaaca actgttacca gttctttgat gagtccaaga actggtacga gtcccaggcc 720 tcttgcatga gccagaatgc ctccctgctg aaggtgtact ctaaggagga ccaggatctg 780 ctgaagctgg tgaagtctta tcactggatg ggcctggtgc acatcccaac caacggcagc 840 tggcagtggg aggacggctc catcctgtct cccaatctgc tgacaatcat cgagatgcag 900 aagggcgatt gtgccctgta tgccagctcc ttcaaagggt atatcgagaa ttgctccact 960 ccaaacactt acatctgtat gcagcggacc gtgaccacga cgccagcgcc gcgaccacca 1020 acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 1080 gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 1140 gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1200 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1260 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1320 gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1380 cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1440 cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1500 tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1560 gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1620 gacacctacg acgcccttca catgcaggcc ctgccccctc gc 1662 <210> 26 <211> 2295 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 26 atggctctgc ccgtgaccgc cctgctgctg cccctggctc tgctgctgca cgccgcccgc 60 cctgtgacaa gacagatgtg catctatacc aaccccacat cctgcaatga gatctacggc 120 aagttcagct ccgcctatct ggcctgtgac ggcaagcaga tggagatcat caccctgctg 180 aacccatctc tgatcagcgg cgatgagtgg cagtggtccg gcaatacacc catccacgtg 240 ctgggcatgt ggcactactc taaggtgctg aagctgctgg accaggatga gaagtcttat 300 gtgaagctgc tgagcgccaa ccagtccatg tgctctgccc agagcgagta ctggaataag 360 tccgaggact tctttcagta ctgcaacaat aagtattgta tctggaacaa gccatgcccc 420 ggctgttatt ctgagacact gcccatcagc ggaggaggag gatccggcgg aggaggctct 480 ggcggcggcg gctccggctc tggaggagga ggatccatcc ctctgacaga gtcttactgc 540 ggcccttgtc caaagaattg gatctgctac aagaacaact gttaccagtt ctttgatgag 600 tccaagaact ggtatgagtc ccaggcctct tgtatgagcc agaatgccag cctgctgaag 660 gtgtactcca aggaggacca ggatctgctg aagctggtga agagctatca ctggatgggc 720 ctggtgcaca tccctaccaa cggctcctgg cagtgggagg acggctccat cctgtctcca 780 aatctgctga caatcatcga gatgcagaag ggcgattgcg ccctgtacgc ctctagcttc 840 aagggctata tcgagaactg cagcacccca aatacataca tctgtatgca gcgcaccgtg 900 accacaaccc cagcacctcg gccccctacc ccagcaccaa caatcgcaag ccagcctctg 960 tccctgcgcc cagaggcatg taggccagca gcaggaggag cagtgcacac cagaggcctg 1020 gactttgcct gcgatatcta tatctgggca cctctggcag gaacctgtgg cgtgctgctg 1080 ctgagcctgg tcatcaccct gtactgcaag agaggcagga agaagctgct gtatatcttc 1140 aagcagccct ttatgcgccc tgtgcagaca acccaggagg aggacggctg ctcctgtagg 1200 ttcccagaag aggaggaggg aggatgtgag ctgagagtga agtttagcag gtccgccgat 1260 gcacctgcat accagcaggg acagaaccag ctgtataacg agctgaatct gggccggaga 1320 gaggagtacg acgtgctgga taagaggagg ggacgggacc ccgagatggg aggcaagcct 1380 cggagaaaga acccacagga gggcctgtac aatgagctgc agaaggacaa gatggccgag 1440 gcctattctg agatcggcat gaagggagag aggcgccggg gcaagggaca cgatggcctg 1500 taccagggcc tgagcaccgc cacaaaggac acctatgatg ccctgcacat gcaggccctg 1560 ccaccaagag gatctggaga gggaaggggc agcctgctga catgcggcga cgtggaggag 1620 aaccctggcc caatgagcag actgccagtg ctgctgctgc tgcagctgct ggtgaggccc 1680 ggcctgcagg cacctatgac ccagacaacc cccctgaaga caagctgggt gaactgttcc 1740 aatatgatcg acgagatcat cacccacctg aagcagcctc cactgcctct gctggatttc 1800 aacaatctga atggcgagga ccaggatatc ctgatggaga acaatctgag aaggccaaac 1860 ctggaggcct ttaatagagc cgtgaagtct ctgcagaacg cctctgccat cgagagcatc 1920 ctgaagaatc tgctgccttg cctgccactg gcaaccgcag caccaacaag gcaccccatc 1980 cacatcaagg acggcgattg gaacgagttc cgccggaagc tgacctttta cctgaagaca 2040 ctggagaatg cccaggccca gcagacaacc ctgagcctgg ccatcttcac aaccacacca 2100 gcacctcgcc ccccaactcc tgccccaaca atcgcatccc agccactgtc tctgcgcccc 2160 gaggcatgca ggcctgcagc aggcggcgcc gtgcacaccc ggggcctgga ctttgcctgt 2220 gatatctaca tctgggcccc cctggccgga acttgtggcg tcctgctgct gtccctggtc 2280 atcactctgt attgc 2295 <210> 27 <211> 1818 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 27 atggctctgc ccgtgactgc cctgctgctg cctctggctc tgctgctgca tgctgctcgc 60 ccaatccctc tgactgaatc atactgtggc ccatgcccca agaactggat ctgctacaag 120 aacaattgtt atcagttctt tgacgagtct aagaactggt acgagtccca ggcctcttgt 180 atgagccaga atgcctctct gctgaaggtg tacagcaagg aggaccagga tctgctgaag 240 ctggtgaaga gctatcactg gatgggcctg gtgcacatcc ccaccaacgg ctcctggcag 300 tgggaggacg gctccatcct gtctcctaat ctgctgacaa tcatcgagat gcagaagggc 360 gattgcgccc tgtacgccag ctccttcaag ggctatatcg agaactgcag caccccaaat 420 acatacatct gtatgcagag gaccgtgacc acaacccctg caccacggcc ccctacccca 480 gcacctacaa tcgcaagcca gccactgtcc ctgcgccccg aggcatgtag gcctgcagca 540 ggcggcgccg tgcacaccag aggcctggac tttgcctgcg atatctatat ctgggcacct 600 ctggcaggaa cctgtggcgt gctgctgctg agcctggtca tcaccctgta ctgcaagaga 660 ggcaggaaga agctgctgta tatcttcaag cagcctttta tgcgcccagt gcagacaacc 720 caggaggagg acggctgctc ttgtcggttc ccagaggagg aggagggcgg ctgtgagctg 780 agagtgaagt tttctaggag cgccgatgca ccagcatacc agcagggaca gaaccagctg 840 tataacgagc tgaatctggg ccggagagag gagtacgacg tgctggataa gaggagggga 900 cgggaccccg agatgggagg caagccacgg agaaagaacc cccaggaggg cctgtacaat 960 gagctgcaga aggacaagat ggccgaggcc tattctgaga tcggcatgaa gggagagagg 1020 cgccggggca agggacacga tggcctgtac cagggcctga gcaccgccac aaaggacacc 1080 tatgatgccc tgcacatgca ggccctgcca ccaagaggat ccggagaggg caggggctct 1140 ctgctgacat gcggcgatgt ggaggagaac ccaggcccca tgtccagact gcctgtgctg 1200 ctgctgctgc agctgctggt gaggcctggc ctgcaggcac caatgaccca gacaacccca 1260 ctgaagacaa gctgggtgaa ctgttccaat atgatcgacg agatcatcac ccacctgaag 1320 cagcctccac tgcccctgct ggatttcaac aatctgaatg gcgaggacca ggatatcctg 1380 atggagaaca atctgagaag gcctaacctg gaggccttta atagagccgt gaagagcctg 1440 cagaacgcct ctgccatcga gagcatcctg aagaatctgc tgccatgcct gccactggca 1500 accgcagcac ccacaaggca ccctatccac atcaaggacg gcgattggaa cgagttccgc 1560 cggaagctga ccttttatct gaagacactg gagaatgccc aggcccagca gacaaccctg 1620 tccctggcca tcttcacaac cacacctgca ccacgccccc caactcctgc ccctacaatc 1680 gcatcccagc cactgtctct gcgccctgag gcatgtcggc cagccgccgg aggagccgtg 1740 cacacccggg gcctggattt cgcttgtgac atctacattt gggctcctct ggctggcacc 1800 tgtggggtcc tgctgctg 1818 <210> 28 <211> 1 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <220> <221> VARIANT <222> (1)...(1) <223> can be present in repeat of at least 1 <400> 28 Gly One <210> 29 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <220> <221> VARIANT <222> (1)...(2) <223> can be present in repeat of at least 1 <400> 29 Gly Ser One <210> 30 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <220> <221> VARIANT <222> (1)...(5) <223> can be present in repeat of at least 1 <400> 30 Gly Ser Gly Gly Ser 1 5 <210> 31 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <220> <221> VARIANT <222> (1)...(4) <223> can be present in repeat of at least 1 <400> 31 Gly Gly Gly Ser One <210> 32 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <220> <221> VARIANT <222> (1)...(5) <223> can be present in repeat of at least 1 <400> 32 Gly Gly Gly Gly Ser 1 5 <210> 33 <211> 516 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 33 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys 20 25 30 Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp 35 40 45 Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn 50 55 60 Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys 65 70 75 80 Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn 85 90 95 Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu 100 105 110 Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser 115 120 125 Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys 130 135 140 Met Gln Arg Thr Val Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 145 150 155 160 Ser Gly Gly Gly Ser Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys 165 170 175 Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp 180 185 190 Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn 195 200 205 Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys 210 215 220 Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn 225 230 235 240 Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu 245 250 255 Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser 260 265 270 Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys 275 280 285 Met Gln Arg Thr Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 290 295 300 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 305 310 315 320 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 325 330 335 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 340 345 350 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 355 360 365 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 370 375 380 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 385 390 395 400 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 405 410 415 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 420 425 430 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 435 440 445 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 450 455 460 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 465 470 475 480 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 485 490 495 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 500 505 510 Leu Pro Pro Arg 515 <210> 34 <211> 523 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 34 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Val Thr Arg Gln Met Cys Ile Tyr Thr Asn Pro 20 25 30 Thr Ser Cys Asn Glu Ile Tyr Gly Lys Phe Ser Ser Ala Tyr Leu Ala 35 40 45 Cys Asp Gly Lys Gln Met Glu Ile Ile Thr Leu Leu Asn Pro Ser Leu 50 55 60 Ile Ser Gly Asp Glu Trp Gln Trp Ser Gly Asn Thr Pro Ile His Val 65 70 75 80 Leu Gly Met Trp His Tyr Ser Lys Val Leu Lys Leu Leu Asp Gln Asp 85 90 95 Glu Lys Ser Tyr Val Lys Leu Leu Ser Ala Asn Gln Ser Met Cys Ser 100 105 110 Ala Gln Ser Glu Tyr Trp Asn Lys Ser Glu Asp Phe Phe Gln Tyr Cys 115 120 125 Asn Asn Lys Tyr Cys Ile Trp Asn Lys Pro Cys Pro Gly Cys Tyr Ser 130 135 140 Glu Thr Leu Pro Ile Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Gly Gly Gly Gly Ser Gly Ser Gly Gly Gly Gly Ser Ile Pro Leu Thr 165 170 175 Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 180 185 190 Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 195 200 205 Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys 210 215 220 Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 225 230 235 240 Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 245 250 255 Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 260 265 270 Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 275 280 285 Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro 290 295 300 Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 305 310 315 320 Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 325 330 335 Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 340 345 350 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 355 360 365 Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 370 375 380 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 385 390 395 400 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 405 410 415 Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 420 425 430 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 435 440 445 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 450 455 460 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 465 470 475 480 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 485 490 495 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 500 505 510 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 515 520 <210> 35 <211> 372 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 35 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys 20 25 30 Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp 35 40 45 Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn 50 55 60 Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys 65 70 75 80 Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn 85 90 95 Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu 100 105 110 Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser 115 120 125 Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys 130 135 140 Met Gln Arg Thr Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 145 150 155 160 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 165 170 175 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 180 185 190 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 195 200 205 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 210 215 220 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 225 230 235 240 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 245 250 255 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 260 265 270 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 275 280 285 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 290 295 300 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 305 310 315 320 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 325 330 335 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 340 345 350 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 355 360 365 Leu Pro Pro Arg 370 <210> 36 <211> 765 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 36 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Val Thr Arg Gln Met Cys Ile Tyr Thr Asn Pro 20 25 30 Thr Ser Cys Asn Glu Ile Tyr Gly Lys Phe Ser Ser Ala Tyr Leu Ala 35 40 45 Cys Asp Gly Lys Gln Met Glu Ile Ile Thr Leu Leu Asn Pro Ser Leu 50 55 60 Ile Ser Gly Asp Glu Trp Gln Trp Ser Gly Asn Thr Pro Ile His Val 65 70 75 80 Leu Gly Met Trp His Tyr Ser Lys Val Leu Lys Leu Leu Asp Gln Asp 85 90 95 Glu Lys Ser Tyr Val Lys Leu Leu Ser Ala Asn Gln Ser Met Cys Ser 100 105 110 Ala Gln Ser Glu Tyr Trp Asn Lys Ser Glu Asp Phe Phe Gln Tyr Cys 115 120 125 Asn Asn Lys Tyr Cys Ile Trp Asn Lys Pro Cys Pro Gly Cys Tyr Ser 130 135 140 Glu Thr Leu Pro Ile Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Gly Gly Gly Gly Ser Gly Ser Gly Gly Gly Gly Ser Ile Pro Leu Thr 165 170 175 Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn 180 185 190 Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln 195 200 205 Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys 210 215 220 Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 225 230 235 240 Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser 245 250 255 Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp 260 265 270 Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser 275 280 285 Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr Pro 290 295 300 Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 305 310 315 320 Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 325 330 335 Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 340 345 350 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 355 360 365 Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 370 375 380 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 385 390 395 400 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 405 410 415 Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 420 425 430 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 435 440 445 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 450 455 460 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 465 470 475 480 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 485 490 495 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 500 505 510 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Glu Gly 515 520 525 Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro 530 535 540 Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gln Leu Leu Val Arg Pro 545 550 555 560 Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp 565 570 575 Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln 580 585 590 Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln 595 600 605 Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe 610 615 620 Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile 625 630 635 640 Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr 645 650 655 Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg 660 665 670 Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 675 680 685 Thr Thr Leu Ser Leu Ala Ile Phe Thr Thr Thr Pro Ala Pro Arg Pro 690 695 700 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 705 710 715 720 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 725 730 735 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 740 745 750 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 755 760 765 <210> 37 <211> 606 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 37 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys 20 25 30 Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp 35 40 45 Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn 50 55 60 Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys 65 70 75 80 Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn 85 90 95 Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu 100 105 110 Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser 115 120 125 Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys 130 135 140 Met Gln Arg Thr Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 145 150 155 160 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 165 170 175 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 180 185 190 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 195 200 205 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 210 215 220 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 225 230 235 240 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 245 250 255 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 260 265 270 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 275 280 285 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 290 295 300 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 305 310 315 320 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 325 330 335 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 340 345 350 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 355 360 365 Leu Pro Pro Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys 370 375 380 Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser Arg Leu Pro Val Leu 385 390 395 400 Leu Leu Leu Gln Leu Leu Val Arg Pro Gly Leu Gln Ala Pro Met Thr 405 410 415 Gln Thr Thr Pro Leu Lys Thr Ser Trp Val Asn Cys Ser Asn Met Ile 420 425 430 Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp 435 440 445 Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn 450 455 460 Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu 465 470 475 480 Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys 485 490 495 Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys 500 505 510 Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys 515 520 525 Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu Ser Leu Ala Ile 530 535 540 Phe Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 545 550 555 560 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 565 570 575 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 580 585 590 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 595 600 605 <210> 38 <211> 1554 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 38 atggctctgc ccgtgactgc cctgctgctg cctctggctc tgctgctgca tgctgctcgc 60 ccaatccctc tgactgaatc atactgtggc ccatgcccca agaactggat ctgctacaag 120 aacaattgtt atcagttctt tgacgagtct aagaactggt acgagtccca ggcctcttgt 180 atgagccaga atgcctctct gctgaaggtg tacagcaagg aggaccagga tctgctgaag 240 ctggtgaaga gctatcactg gatgggcctg gtgcacatcc ccaccaacgg ctcctggcag 300 tgggaggacg gctccatcct gtctcctaat ctgctgacaa tcatcgagat gcagaagggc 360 gattgcgccc tgtacgccag ctccttcaag ggctatatcg agaactgcag caccccaaat 420 acatacatct gtatgcagag gaccgtggga ggaggaagcg gaggaggatc cggaggcggc 480 tctggcggcg gcagcatccc tctgactgaa tcatactgtg gcccatgccc caagaactgg 540 atctgctaca agaacaattg ttatcagttc tttgacgagt ctaagaactg gtacgagtcc 600 caggcctctt gtatgagcca gaatgcctct ctgctgaagg tgtacagcaa ggaggaccag 660 gatctgctga agctggtgaa gagctatcac tggatgggcc tggtgcacat ccccaccaac 720 ggctcctggc agtgggagga cggctccatc ctgtctccta atctgctgac aatcatcgag 780 atgcagaagg gcgattgcgc cctgtacgcc agctccttca agggctatat cgagaactgc 840 agcaccccaa atacatacat ctgtatgcag aggaccgtga ctagtaccac gacgccagcg 900 ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag 960 gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgcctgtgat 1020 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1080 accctttact gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1140 agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1200 gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtaccag 1260 cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1320 ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1380 caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1440 gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1500 acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgc 1554 <210> 39 <211> 1569 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 39 atggctctgc ccgtgaccgc cctgctgctg cccctggctc tgctgctgca cgccgcccgc 60 cctgtgacaa gacagatgtg catctatacc aaccccacat cctgcaatga gatctacggc 120 aagttcagct ccgcctatct ggcctgtgac ggcaagcaga tggagatcat caccctgctg 180 aacccatctc tgatcagcgg cgatgagtgg cagtggtccg gcaatacacc catccacgtg 240 ctgggcatgt ggcactactc taaggtgctg aagctgctgg accaggatga gaagtcttat 300 gtgaagctgc tgagcgccaa ccagtccatg tgctctgccc agagcgagta ctggaataag 360 tccgaggact tctttcagta ctgcaacaat aagtattgta tctggaacaa gccatgcccc 420 ggctgttatt ctgagacact gcccatcagc ggaggaggag gatccggcgg aggaggctct 480 ggcggcggcg gctccggctc tggaggagga ggatccatcc ctctgacaga gtcttactgc 540 ggcccttgtc caaagaattg gatctgctac aagaacaact gttaccagtt ctttgatgag 600 tccaagaact ggtatgagtc ccaggcctct tgtatgagcc agaatgccag cctgctgaag 660 gtgtactcca aggaggacca ggatctgctg aagctggtga agagctatca ctggatgggc 720 ctggtgcaca tccctaccaa cggctcctgg cagtgggagg acggctccat cctgtctcca 780 aatctgctga caatcatcga gatgcagaag ggcgattgcg ccctgtacgc ctctagcttc 840 aagggctata tcgagaactg cagcacccca aatacataca tctgtatgca gcgcaccgtg 900 accacaaccc cagcacctcg gccccctacc ccagcaccaa caatcgcaag ccagcctctg 960 tccctgcgcc cagaggcatg taggccagca gcaggaggag cagtgcacac cagaggcctg 1020 gactttgcct gcgatatcta tatctgggca cctctggcag gaacctgtgg cgtgctgctg 1080 ctgagcctgg tcatcaccct gtactgcaag agaggcagga agaagctgct gtatatcttc 1140 aagcagccct ttatgcgccc tgtgcagaca acccaggagg aggacggctg ctcctgtagg 1200 ttcccagaag aggaggaggg aggatgtgag ctgagagtga agtttagcag gtccgccgat 1260 gcacctgcat accagcaggg acagaaccag ctgtataacg agctgaatct gggccggaga 1320 gaggagtacg acgtgctgga taagaggagg ggacgggacc ccgagatggg aggcaagcct 1380 cggagaaaga acccacagga gggcctgtac aatgagctgc agaaggacaa gatggccgag 1440 gcctattctg agatcggcat gaagggagag aggcgccggg gcaagggaca cgatggcctg 1500 taccagggcc tgagcaccgc cacaaaggac acctatgatg ccctgcacat gcaggccctg 1560 ccaccaaga 1569 <210> 40 <211> 1122 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 40 atggctctgc ccgtgactgc cctgctgctg cctctggctc tgctgctgca tgctgctcgc 60 ccaatccctc tgactgaatc atactgtggc ccatgcccca agaactggat ctgctacaag 120 aacaattgtt atcagttctt tgacgagtct aagaactggt acgagtccca ggcctcttgt 180 atgagccaga atgcctctct gctgaaggtg tacagcaagg aggaccagga tctgctgaag 240 ctggtgaaga gctatcactg gatgggcctg gtgcacatcc ccaccaacgg ctcctggcag 300 tgggaggacg gctccatcct gtctcctaat ctgctgacaa tcatcgagat gcagaagggc 360 gattgcgccc tgtacgccag ctccttcaag ggctatatcg agaactgcag caccccaaat 420 acatacatct gtatgcagag gaccgtgact agtaccacga cgccagcgcc gcgaccacca 480 acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 540 gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 600 gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 660 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 720 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 780 gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 840 cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 900 cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 960 tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1020 gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1080 gacacctacg acgcccttca catgcaggcc ctgccccctc gc 1122 <210> 41 <211> 221 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 41 Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gln Leu Leu Val Arg Pro 1 5 10 15 Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp 20 25 30 Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln 35 40 45 Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln 50 55 60 Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe 65 70 75 80 Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile 85 90 95 Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr 100 105 110 Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg 115 120 125 Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 130 135 140 Thr Thr Leu Ser Leu Ala Ile Phe Thr Thr Thr Pro Ala Pro Arg Pro 145 150 155 160 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 165 170 175 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 180 185 190 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 195 200 205 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 210 215 220 <210> 42 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 42 Met Ser Arg Leu Pro Val Leu Leu Leu Leu Gln Leu Leu Val Arg Pro 1 5 10 15 Gly Leu Gln Ala Pro Met Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp 20 25 30 Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln 35 40 45 Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln 50 55 60 Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe 65 70 75 80 Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile 85 90 95 Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr 100 105 110 Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg 115 120 125 Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln 130 135 140 Thr Thr Leu Ser Leu Ala Ile Phe Thr Thr Thr Pro Ala Pro Arg Pro 145 150 155 160 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 165 170 175 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 180 185 190 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 195 200 205 Gly Val Leu Leu Leu 210 <210> 43 <211> 663 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 43 atgagcagac tgccagtgct gctgctgctg cagctgctgg tgaggcccgg cctgcaggca 60 cctatgaccc agacaacccc cctgaagaca agctgggtga actgttccaa tatgatcgac 120 gagatcatca cccacctgaa gcagcctcca ctgcctctgc tggatttcaa caatctgaat 180 ggcgaggacc aggatatcct gatggagaac aatctgagaa ggccaaacct ggaggccttt 240 aatagagccg tgaagtctct gcagaacgcc tctgccatcg agagcatcct gaagaatctg 300 ctgccttgcc tgccactggc aaccgcagca ccaacaaggc accccatcca catcaaggac 360 ggcgattgga acgagttccg ccggaagctg accttttacc tgaagacact ggagaatgcc 420 caggcccagc agacaaccct gagcctggcc atcttcacaa ccacaccagc acctcgcccc 480 ccaactcctg ccccaacaat cgcatcccag ccactgtctc tgcgccccga ggcatgcagg 540 cctgcagcag gcggcgccgt gcacacccgg ggcctggact ttgcctgtga tatctacatc 600 tgggcccccc tggccggaac ttgtggcgtc ctgctgctgt ccctggtcat cactctgtat 660 tgc 663 <210> 44 <211> 639 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 44 atgtccagac tgcctgtgct gctgctgctg cagctgctgg tgaggcctgg cctgcaggca 60 ccaatgaccc agacaacccc actgaagaca agctgggtga actgttccaa tatgatcgac 120 gagatcatca cccacctgaa gcagcctcca ctgcccctgc tggatttcaa caatctgaat 180 ggcgaggacc aggatatcct gatggagaac aatctgagaa ggcctaacct ggaggccttt 240 aatagagccg tgaagagcct gcagaacgcc tctgccatcg agagcatcct gaagaatctg 300 ctgccatgcc tgccactggc aaccgcagca cccacaaggc accctatcca catcaaggac 360 ggcgattgga acgagttccg ccggaagctg accttttatc tgaagacact ggagaatgcc 420 caggcccagc agacaaccct gtccctggcc atcttcacaa ccacacctgc accacgcccc 480 ccaactcctg cccctacaat cgcatcccag ccactgtctc tgcgccctga ggcatgtcgg 540 ccagccgccg gaggagccgt gcacacccgg ggcctggatt tcgcttgtga catctacatt 600 tgggctcctc tggctggcac ctgtggggtc ctgctgctg 639 <210> 45 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 45 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15

Claims (30)

Chimeric receptors comprising the following polypeptide chains:
(a) an extracellular domain comprising an NKG2D domain;
(b) transmembrane domain; And
(c) Intracellular signaling domain. Multispecific chimeric receptors comprising the following polypeptide chains:
(a) an extracellular domain comprising a first NKG2D domain, a second NKG2D domain, and a second antigen binding domain;
(b) transmembrane domain; And
(c) Intracellular signaling domain. The multispecific chimeric receptor according to claim 2, wherein the extracellular domain is in the N-terminal to C-terminal direction, comprising: a second antigen binding domain, a first NKG2D domain and a second NKG2D domain. The multispecific chimeric receptor according to any one of claims 1 to 3, wherein the second antigen binding domain is fused to the first NKG2D domain through a peptide linker. A multispecific chimeric receptor comprising a first polypeptide chain and a second polypeptide chain, wherein each chain comprises:
(a) an extracellular domain including an NKG2D domain and a second antigen binding domain;
(b) transmembrane domain; And
(c) Intracellular signaling domain. The multispecific chimeric receptor of claim 5, wherein each extracellular domain further comprises a dimerization motif. The multispecific chimeric receptor of claim 6, wherein the dimerization motif is replaced between the NKG2D domain and the second antigen binding domain. The multispecific chimeric receptor according to claim 6 or 7, wherein the dimerization motif is a leucine zipper. The multispecific chimeric receptor of claim 8, wherein the second antigen binding domain is fused to the first NKG2D domain through a peptide linker. The multispecific chimeric receptor according to any one of claims 5 to 9, wherein each of the first polypeptide chain and the second polypeptide chain from N-terminus to C-terminus comprises: second antigen binding domain, NKG2D domain. , A transmembrane domain, and the intracellular signaling domain. Dual chimeric receptor system, including:
(i) a first chimeric receptor comprising:
(a) an extracellular domain comprising a first NKG2D domain;
(b) a first transmembrane domain; And
(c) a first intracellular signaling domain; And
(ii) a second chimeric receptor comprising:
(a) a second extracellular domain comprising a second antigen binding domain; And
(b) a second transmembrane domain. The dual chimeric receptor system of claim 11, wherein the second chimeric receptor further comprises a second intracellular signaling domain. The multispecific chimeric receptor, or dual chimeric receptor system of any one of claims 2 to 10, or of claim 11 or 12, wherein the second antigen binding domain is an antibody fragment. The method of claim 13, wherein the antibody fragment is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, GD-2, NY- A multispecific chimeric receptor, or a dual chimeric receptor system that specifically binds to an antigen selected from the group consisting of ESO-1, MAGE A3, and glycolipid F77. The multispecific chimeric receptor, or dual chimeric receptor system of any one of claims 2 to 10, or claim 11 or 12, wherein the second antigen binding domain is a ligand or a ligand binding domain. The method of claim 15, wherein the ligand or ligand binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. Chimeric receptor, or dual chimeric receptor system. The multispecific chimeric receptor system of claim 16, wherein the second antigen binding domain is an IL-3 domain. The method of any one of claims 1 to 10 and 13 to 17, or any one of claims 11 to 17, wherein the NKG2D domain (or the first NKG2D domain and/or the second NKG2D domain) is SEQ ID NO: 7 or A chimeric receptor, or a dual chimeric receptor system comprising the amino acid sequence of 8. The method according to any one of claims 1 to 10 and 13 to 18, or any one of claims 11 to 18, wherein the transmembrane domain (or the first transmembrane domain and/or the second transmembrane domain) is CD8α, A chimeric receptor, or a dual chimeric receptor system, derived from a molecule selected from the group consisting of CD4, CD28, 4-1BB, CD80, CD86, CD152 and PD1. The method according to any one of claims 1 to 10 and 13 to 19, or any one of claims 11 to 19, wherein the intracellular signaling domain (or the first intracellular signaling domain and/or the second intracellular Signaling domain) is a chimeric receptor, or a dual chimeric receptor system, comprising the primary intracellular signaling domain of an immune effector cell. The method of any one of claims 1 to 10 and 13 to 20, or any one of claims 11 to 20, wherein the intracellular signaling domain (or the first intracellular signaling domain and/or the second intracellular The signaling domain) comprises a co-stimulatory signaling domain, a chimeric receptor, or a dual chimeric receptor system. The method of claim 21, wherein the co-stimulatory signaling domain is CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. A chimeric receptor, or a dual chimeric receptor system, derived from a co-stimulatory molecule selected from the group consisting of ligands and combinations thereof. A chimeric receptor comprising an amino acid sequence having at least about 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-20 and 33-35. A polypeptide comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 36 or 37. An isolated nucleic acid comprising a nucleic acid sequence encoding a chimeric receptor according to any one of claims 1 to 10 and 13 to 23. The method of any one of claims 11 to 22, wherein the isolated nucleic acid comprises a first nucleic acid sequence encoding a first chimeric receptor and a second nucleic acid sequence encoding a second chimeric receptor, wherein the first nucleic acid sequence is self- An isolated nucleic acid operably linked to a second nucleic acid sequence via a third nucleic acid sequence encoding a cleavage peptide. An isolated nucleic acid comprising a nucleic acid sequence having at least about 85% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 21-27 and SEQ ID NOs: 38-40. An engineered immune effector cell comprising the chimeric receptor or dual chimeric receptor system according to any one of claims 1 to 23, or the isolated nucleic acid according to any one of claims 25 to 27. A pharmaceutical composition comprising an engineered immune effector cell according to claim 28, and a pharmaceutically acceptable carrier. A method of treating cancer in a subject, wherein the method comprises administering to the subject an effective amount of a pharmaceutical composition according to claim 29.

Images